Elsevier

Molecular Genetics and Metabolism

Volume 110, Issues 1–2, September–October 2013, Pages 116-121
Molecular Genetics and Metabolism

Differences between acylcarnitine profiles in plasma and bloodspots

https://doi.org/10.1016/j.ymgme.2013.04.008Get rights and content

Highlights

  • Acylcarnitines profiles in plasma and DBS were measured simultaneously.

  • Natural levels of acylcarnitines differ between plasma and DBS.

  • The choice of matrix for acylcarnitine profiling is critical for diagnostics.

  • To detect CPT-2 deficiency, plasma is the better matrix.

Abstract

Quantification of acylcarnitines is used for screening and diagnosis of inborn error of metabolism (IEM). While newborn screening is performed in dried blood spots (DBSs), general metabolic investigation is often performed in plasma. Information on the correlation between plasma and DBS acylcarnitine profiles is scarce. In this study, we directly compared acylcarnitine concentrations measured in DBS with those in the corresponding plasma sample. Additionally, we tested whether ratios of acylcarnitines in both matrices are helpful for diagnostic purpose when primary markers fail.

Study design

DBS and plasma were obtained from controls and patients with a known IEM. (Acyl)carnitines were converted to their corresponding butyl esters and analyzed using HPLC/MS/MS.

Results

Free carnitine concentrations were 36% higher in plasma compared to DBS. In contrast, in patients with carnitine palmitoyltransferase 1 (CPT-1) deficiency free carnitine concentration in DBS was 4 times the concentration measured in plasma. In carnitine palmitoyltransferase 2 (CPT-2) deficiency, primary diagnostic markers were abnormal in plasma but could also be normal in DBS. The calculated ratios for CPT-1 (C0/(C16 + C18)) and CPT-2 ((C16 + C18:1)/C2) revealed abnormal values in plasma. However, normal ratios were found in DBS of two (out of five) samples obtained from patients diagnosed with CPT-2.

Conclusions

Relying on primary acylcarnitine markers, CPT-1 deficiency can be missed when analysis is performed in plasma, whereas CPT-2 deficiency can be missed when analysis is performed in DBS. Ratios of the primary markers to other acylcarnitines restore diagnostic recognition completely for CPT-1 and CPT-2 in plasma, while CPT-2 can still be missed in DBS.

Introduction

As the mitochondrial membrane is impermeable to long chain fatty acids, the carnitine shuttle is used to import acyl-CoA's. Acyl-CoA's can cross the mitochondrial membrane via carnitine acylcarnitine translocase (CACT) after conversion to acylcarnitines by carnitine-palmitoyl CoA transferase 1 (CPT-1). Reconversion of acylcarnitines to acyl-CoA's by carnitine-palmitoyl CoA transferase 2 (CPT-2) provides very-long chain acyl-CoA dehydrogenase (VLCAD) with the degradable acyl-CoA's to ensure energy supply. In addition, potentially toxic acyl-CoA's can be removed via the same route. Accumulation of specific acyl-CoA's due to a metabolic block leaves the cell as acylcarnitines [1], [2].

In body fluids, the acylcarnitine profile is not only a diagnostic test for inherited disorders of fatty acid metabolism, but also for defects in branched-chain amino acid catabolism [2]. Patients with these types of metabolic disorders accumulate disease-specific acylcarnitines, since degradation of amino acids produces, in many cases, odd-chain acyl-compounds that are esterified with carnitine.

While in biochemical genetic laboratories plasma is routinely used for acylcarnitine analysis, newborn screening programs use whole blood dried on filter paper (DBS) as the standard specimen. Newborns who show abnormal screening results are referred to the clinical unit for diagnosis and treatment. The workflow in our department primarily involves confirmation by biochemical testing (by measurement of plasma acylcarnitine profile) followed by additional tests (e.g. enzymatic assays or DNA mutation analysis). While cut-off points for free carnitine and acylcarnitine esters have been published for both DBS [3], [4] and plasma [5], [6] only limited information is available on the correlation between plasma and DBS.

Comparison between free carnitine in plasma and DBS from patients with organic acidurias and fatty acid oxidation disorders were reported by Primassin and Spiekerkoetter [7]. Data on comparison between acylcarnitine concentrations in the different matrices is scarce.

The use of absolute concentrations may lead to be potential interpretative problems. In newborn screening programs several ratios between different acylcarnitines have been reported that could help as a discriminate factor [4]. Such information is widely available for DBS but only limited for plasma.

This study examines acylcarnitines profiles in plasma and DBS simultaneously in samples from patients with well-defined inborn errors of metabolism (IEM). Subsequently, we evaluated whether ratios of acylcarnitines in plasma are just as helpful as these ratios are in DBS when primary markers fail to be conclusive.

Section snippets

Study population

Blood was collected (for therapeutic control) from patients with confirmed (enzymatic or molecular) inherited metabolic diseases. These included plasma and DBS from patients diagnosed with different enzyme or transporter deficiencies: CPT-1 deficiency (n = 6 samples, 2 patients), CPT-2 deficiency (n = 5 samples, 4 patients), VLCAD deficiency (n = 12 samples, 11 patients), long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency (n = 2), and medium-chain acyl-CoA dehydrogenase (MCAD) deficiency (n = 

Results

Fig. 1 shows representative acylcarnitine profiles in DBS and plasma from a neonatal control subject. When focusing on individual acylcarnitines, significant differences between plasma and DBS were observed (Table 1). In general, free carnitine concentrations were 36% higher in plasma compared to free carnitine concentrations in DBS (Table 1; Fig. 2; P < 0.0001). Plasma C8 carnitine concentration was twice the concentration measured in DBS (for controls as well as patients diagnosed with MCAD

Discussion

HPLC/MS/MS is the method of choice for the quantification of (acyl)carnitines in body fluids. While it is known that carnitine concentrations in tissues are normally 20- to 50-fold higher than in serum [8], there is no consensus concerning the clinical specimen (plasma or DBS) that would best reflect the carnitine status of various body compartments. DBS offers a number of advantages over conventional plasma collection. DBS is preferred since it requires a less invasive sampling method (finger

Conflict of interest

The authors wish to confirm that there are no known conflicts of interest associated with this publication and there was no financial support for this work.

Acknowledgments

We would like to thank Karen van Baal for performing HPLC/MS/MS experiments.

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