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Laboratory Management |
1 Newborn Screening Center, Laboratory of Pediatrics, Free University of Brussels, Brussels, Belgium.
2 Scientific Institute for Public Health (IPH), Epidemiology Unit, Brussels, Belgium.
3 Department of Pediatrics, Queen Fabiola University Children’s Hospital, Free University of Brussels, Brussels, Belgium.
aAddress correspondence to this author at: 80 Strathcona Gardens, G13 1DN, Glasgow, United Kingdom. Fax 44-32-2-477-2563; e-mail catia.cavedon{at}gmail.com.
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Abstract |
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Methods: Filter-paper blood spots were collected from 433 healthy individuals over a period of 17 months. Eight age groups were defined: cord blood, 3–6 days (control group), 15–55 days, 2–18 months, 19–59 months, 5–10 years, 11–17 years, and 18–54 years. Free carnitine and acylcarnitines were measured for each individual. Mean values were calculated for each age group and compared with those for the control group.
Results: Free carnitine was significantly higher in older children than in newborns (P <0.05), but the concentrations of several acylcarnitines tended to be significantly lower in cord blood and in groups of older children than in the control group. Only minor sex-related differences were observed.
Conclusion: Although the risk of underdiagnosis of fatty acid oxidation disorders with the use of newborn values as reference can be considered as small, in some circumstances the use of age-related reference values may have a potential impact on the diagnosis and management of inherited errors of metabolism.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reference values for FC and AC profiles by MS/MS have been calculated from the whole-blood concentrations measured in healthy newborns, a period of intense fatty acid metabolism (7)(8). Because the concentration of metabolites such as amino acids and organic acids can change markedly with age, variations in FC and AC concentrations could have a potential impact on the accuracy of the diagnosis and optimal management of patients with IEM. In this study, we measured FC and AC concentrations by MS/MS in dried blood samples obtained from birth to adulthood.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The control group corresponded to the classic neonatal screening group: 117 dried-blood-spot samples received at the Newborn Screening Center on one single, randomly selected day were used to compute mean values for the control group. Premature infants (<37 weeks), infants with a low birth weight (<2500 g), and newborns younger than 3 days or older than 6 days were excluded from the control group. The FC and AC values in this group were not significantly different from reference values obtained from a population of 10 000 newborns previously tested in our laboratory during 2002 (our unpublished data).
Group A (cord blood), which should reflect the situation of the fetus, comprised 67 umbilical cord blood samples collected on filter paper. These newborns (
36 weeks of gestational age) were considered appropriate for gestational age, and their birth weight ranged from 2500 to 4150 g. Of the 67 infants, 60 had been delivered vaginally. The remaining had been delivered by cesarean section, which was indicated for breech presentation and cephalopelvic disproportion. All mothers had a normal outcome. None of these infants presented with signs of severe hypoxia during delivery. The Apgar scores of these newborns ranged from 6 to 10 at the first minute of life. All newborns also had a favorable outcome, without complications until discharge.
Group B (15–55 days; n = 30) corresponded to the "full lactation" group, i.e., receiving frequent feedings (6–8 bottles/day) and presenting with rapid growth. Group C (2–18 months; n = 17) corresponded to the period of diversification of diet, reduction in the number of meals (4–5/day), and lengthening of the overnight fast. Group D (19–59 months; n = 51) corresponded to preschool age, group E (5–10 years; n = 70) to school age, and group F (11–17 years; n = 32) to adolescence (rapid growth).
For groups B through F, samples were collected simultaneously with routine preoperative blood tests from healthy children who had minor elective surgical procedures, except for 16 children attending an elementary school. For children to be eligible for the study, parents were required to respond to a questionnaire. Of a total of 207 questionnaires, only 200 were analyzed because 7 children were later found to be on medication (antibiotics) or special diets, and/or had a diagnosis of chronic disease.
Group G (18–54 years; n = 49) corresponded to adulthood. Adult volunteers were recruited among the hospital staff. Each volunteer was also requested to answer a questionnaire. Blood was not collected from volunteers identified as having chronic and/or acute health problems or who were on medications. The questionnaire did not include information about smoking. The mean (SD) body mass index for this group was 21.88 (2.99) kg/m2.
ethics approval and consent
The study was approved by the Ethical Committee of the Queen Fabiola University Children’s Hospital. Written informed consent was obtained from parents of children under the age of 18 years except for the control group (systematic newborn screening). In addition, personal consent was obtained from older children.
solvents, reagents, and internal standards
High-purity-grade methanol (Methanol Chroma) was obtained from Riedel-de-Haën. Butanolic HCl (3 mol/L) was obtained from Regis Technologies. Acetonitrile (LiChro solvent grade) and formic (98–100%) acid were obtained from Merck. Stable isotopes used as internal standards were obtained from Cambridge Isotope Laboratories and included [2H9]FC, [2H3]acetylcarnitine (C2), [2H3]propionylcarnitine (C3), [2H3]butyrylcarnitine (C4), [2H3]isovalerylcarnitine (C5), [2H9]octanoylcarnitine (C8), [2H3]myristoylcarnitine (C14), and [2H3]palmitoylcarnitine (C16).
sample preparation and analytical procedure
All samples (whole blood) were collected on Schleicher & Schuell Grade 903 filter paper and analyzed by a previously described electrospray-MS/MS method (9)(10), with slight modifications. This filter paper meets NCCLS and CDC specifications.
The analytical procedure is based on butyl ester derivatives of amino acids and ACs. This is the classic procedure used by many newborn-screening centers. Briefly, single disks are punched from each dried blood spot with a standard 3-mm puncher. The different analytes (FC and AC) are extracted from each sample for 30 min at room temperature in 96-well microplates with 200 µL of methanol (Chromasolv grade) containing a mixture of the respective stable-isotope-labeled internal standards (Cambridge Isotopes). For ACs, the internal standards includes deuterated free carnitine (C0), C2, C3, C4, C5, C8, C14, and C16. The solution is then evaporated to dryness on a heating block at 55 °C under nitrogen. Butanolic HCl (50 µL of a 3 mol/L solution) is added to each sample and incubated for 20 min at 65 °C. After incubation, the solution is again evaporated to dryness on a heating block at 55 °C under nitrogen. Derivatized samples are then reconstituted with 200 µL of a mobile phase (acetonitrile–water, 50:50 by volume) and injected for MS/MS analysis at a flow rate of 20 µL/min.
ms analyses
For our analyses, we used an API 365 triple quadrupole tandem mass spectrometer (Perkin-Elmer Sciex) with an ion spray source. Samples were introduced through the tandem mass spectrometer by the atmospheric pressure ionization (API) system, in which different analytes and their internal standards were recognized by sorting their respective mass-to-charge ratio (m/z). We calculated the FC and AC concentrations by comparing the ion mass spectra of the different analytes with the spectra of their corresponding internal standards. MS/MS was carried out as described previously in detail (9)(10)(11), with slight modifications.
statistical analysis
For all analytes, means of age groups were calculated by an ANOVA model that included, in addition to age group, the age of the patient. We used the Tukey test procedure to test differences between the control age group mean and all age group means (12). Calculations were performed with the SAS ANOVA procedure (12). Statistical significance was defined as a P value <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 2
, and 3
.
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fc and short-chain ACS
Althought FC concentrations in groups A and B did not differ significantly from the FC concentration for the control group, mean FC concentrations were significantly higher in groups C through G (Table 1
).
Although the short-chain AC concentrations [C2, malonylcarnitine (C3:DC), C4, hydroxybutyrylcarnitine (C4OH), and glutarylcarnitine (C5:DC)] were considerably lower in group A than in the control group, the differences between group A and the control group were not statistically significant for methylmalonylcarnitine (C4:DC), hydroxyisovalerylcarnitine (C5OH), and (C5). However, the mean C3 values were significantly higher in group A than in the control group.
The C2, C3, C4, and C4OH concentrations were significantly lower in groups B through F compared with the control group. In group B, C4:DC and C5OH were significantly higher than in the control group. The C3:DC, C5:DC, and C5 concentrations were similar between each of the different age groups and the control group. The short-chain AC concentrations in group G were significantly lower than in the control group for C2, C3:DC, C4, C4OH, C4:DC, and C5:DC, whereas C4:DC and C5OH were significantly higher than in the control group.
medium-chain ACS
The medium-chain AC concentrations in group A were significantly lower than in the control group, with the exception of decenoylcarnitine (C10:1). In groups C through G, hexanoylcarnitine (C6), methylglutarylcarnitine (C6:DC), and dodecanoylcarnitine (C12) concentrations were significantly lower than in the control group (Table 2
).
long-chain ACS
In group A, the concentrations of the long-chain ACs C14, tetradecenoylcarnitine (C14:1), C16, hydroxyoctadecanoylcarnitine (C18OH), octadecenoylcarnitine (C18:1), hydroxyoleylcarnitine (C18:1OH), and octadecadienoylcarnitine (C18:2) were significantly lower than in the control group. The tetradecadienoylcarnitine (C14:2), hydroxytetradecanoylcarnitine (C14OH), and hydroxypalmitoylcarnitine (C16OH) concentrations in group A were not significantly different from the control group (Table 3
).
As for medium-chain ACs, the concentrations of the long-chain ACs were lower in groups B through G than in the control group. Although the mean C16OH, C18:1OH, and C18:2 values were significantly lower in group B than in the control group, they were similar for groups C through G. Finally, we found no differences between C14:2 and C14OH concentrations in any of the age groups and the control group.
sex-related differences
Apart from C14, C16, and C18:1 concentrations (results not shown), no other sex-related differences were noted.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our study, FC concentrations were significantly higher in older children than in newborns (control group), but we observed no significant differences between FC concentrations in cord blood and in this same control group. On the other hand, the concentrations of several ACs tended to be significantly lower in cord blood and in groups of older children than in the control group. Only minor sex-related differences were observed in this study.
The comparison of absolute values found in different studies can be complicated by the fact that, in most studies, FC and AC concentrations were measured in plasma samples rather than whole blood and by methods other than MS/MS (15)(16)(17)(18)(19). Despite these differences, some of these studies have also shown that plasma FC concentrations were higher in older children than in newborns. In one study, FC concentrations in males were significantly higher than in females (19).
Some interesting comparisons can be made between our results and those of other studies, in which the whole-blood FC values were also measured by MS/MS. Meyburg et al. (11), who assessed FC values in newborns, found no differences between FC concentrations in cord blood and 5-day-old infants, and the absolute values reported in that study were similar to our data [30.6 (10.6) µmol/L vs 29.4 (1.8) µmol/L]. In another large study (7), absolute FC values in newborns were higher than those observed in our study [46.41 (20.73) µmol/L]. Although this study investigated more than 20 000 full-term newborns, no comparisons were made among different age groups. In our smaller study, we compared FC values among a homogeneous population of Belgian children and performed all assessments at the same laboratory with an identical methodology, thus minimizing the risks of bias.
The lower AC concentrations found in cord blood (compared with the control group) in our study are similar to those reported previously by Meyburg et al. (11). Contrary to the results reported by Takiyama et al. (19), who found similar serum plasma AC concentrations through the different ages (using a different method of assessment), we found significant variation in AC concentrations across the different age groups. Our results are also in agreement with data published by Chace et al. (10), who measured AC by MS/MS in blood spots of 16 newborns and 16 older patients with confirmed medium-chain acyl coenzyme A dehydrogenase (MCAD) deficiency and found significantly higher concentrations of medium-chain ACs in newborns than in older patients. These same authors suggested that these differences might be attributable to either sample degradation (related to storage of samples from older patients with MCAD deficiency) or renal loss. Nevertheless, we observed the same significant differences between newborns and older children, using samples taken from healthy individuals with no delay between sample collection and the analyses.
In clinical practice, it can be speculated that age-related AC reference values could be meaningful in situations in which the diagnostic cutoff of a metabolic disorder is very close to the reference interval. This may well be the case for methylmalonic aciduria, which may occasionally present with only slight increases in short-chain AC concentrations. Other examples are long-chain acyl CoA dehydrogenase and trifunctional protein deficiencies, in which the diagnosis typically is suggested by modest increases in long-chain ACs (14). In our study, concentrations of long-chain ACs were significantly lower in cord blood and in older children than in the control group. This suggests, at least theoretically, that the diagnosis of these conditions could be missed if the concentrations measured in healthy newborns were used as reference values for older children. This is well illustrated by the C16 values, which in most study groups were more than 50% lower than those in the control group. Similarly, AC cord blood concentrations may also be inappropriate for the diagnosis of such fatty acid oxidation disorders. Furthermore, there is a potential risk of undertreatment when older children with metabolic disorders are managed based on newborn reference values.
Age-related AC reference values could also be helpful in other circumstances, such as the investigation of siblings (children with MCAD deficiency who are in stable condition). In this situation, the increase in AC concentrations may be very subtle. Typically, clinical decompensation occurs only when patients are exposed to a metabolic stress (e.g., severe illness, prolonged fasting, and surgery); otherwise, a diagnosis is unlikely to be established (20)(21). In addition, biochemical abnormalities in older children can be hard to diagnose if the patient is in a fed state and healthy.
The cutoffs (reference intervals) for the "age of 5 days" used in our laboratory are shown in Tables 1
, 2
, and 3
. These cutoffs (5th and 95th centiles) are based on our personal experience with 10 000 "day-5" samples previously analyzed at our laboratory (unpublished data). The procedure to measure FC and ACs in this study was exactly the same, but only AC mean (50th centile) concentrations are shown for each age group.
Conversely, in MCAD deficiency, for example, the concentrations in the pathologic state tend to be markedly higher than the reference interval (14). This is also the case of other IEM, such as propionic aciduria, carnitine palmitoyl transferase deficiency II, and isovaleric acidurias, which are also associated with marked increases in the respective ACs (10)(14). Therefore, the statistically significant but small differences in some AC concentrations seen in our study are unlikely to affect the diagnosis of any of these conditions.
It has been demonstrated that FC and AC values are significantly higher in whole blood than in plasma (7)(15)(22) because of the significantly higher concentrations of long-chain ACs in the erythrocyte membrane. Plasma AC concentrations would most likely show a pattern of variation similar to the one observed in whole blood samples. This specific question deserves further studies.
In conclusion, few sex-related differences were observed in our study. Nevertheless, the analysis of whole blood samples by MS/MS disclosed significant age-related variations in AC and FC concentrations. The magnitude of these variations, although statistically significant, was generally small. Although these small variations are unlikely to have a significant impact on the diagnosis of most IEM, they could be useful in a few conditions where the biochemical abnormalities are subtle.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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