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Letters to the Editor |
1 Department of Psychiatry, and Psychotherapy, University of Erlangen-Nuremberg, Erlangen, Germany
2 Center for, Clinical Neuroproteomics, Erlangen, Germany
3 Department of Psychiatry, and Psychotherapy, University of Goettingen, Goettingen, Germany
aAddress correspondence to this author at: Molecular Neurobiology Laboratory, Department of Psychiatry and Psychotherapy, University of Erlangen-Nuremberg, Schwabachanlage 6, 91054 Erlangen, Germany. Fax 49-0-9131-8536381; e-mail Jens.Wiltfang{at}psych.imed.uni-erlangen.de.
To the Editor:
Tau protein and its phosphorylated forms, and amyloid ß peptides ending at amino acid 42 (Aß42) are used as cerebrospinal fluid (CSF) biomarkers of Alzheimer disease (AD) (1)(2)(3)(4)(5)(6)(7)(8). Because preanalytical factors may affect results (4)(9)(10), we measured these biomarkers in CSF samples in collection tubes made of different materials.
After approval by the Ethics Committee of the University of Erlangen-Nuremberg, patients and/or their closest relatives gave written informed consent. CSF was obtained from patients (n = 20; mean age, 66 years; 9 women and 11 men) with various neuropsychiatric diseases, including dementias (n = 9), mild cognitive impairment (n = 8), recurrent depression (n = 1), bipolar disorder (n = 1), and emotionally unstable personality (n = 1). We included only CSF samples showing no or only minor contamination with erythrocytes (<150/µL), no intrathecal humoral or cellular immune response, and normal or only slightly disturbed blood–CSF barrier as measured by albumin quotient. After collecting 4–5 mL of CSF for routine analyses, we transferred four 0.5-mL fractions directly into 4 test tubes in this order: (a), polycarbonate (PC; Sarstedt; cat. no. 60.9922.936); (b), a copolymer of polystyrene and acrylonitrile [modified polystyrene (PX); Sarstedt; cat. no. 60.9924.952]; (c), polystyrene (PS; Sarstedt; cat. no. 62.553.542PS); and (d), polypropylene (PP; Sarstedt; cat. no. 62.554.502PP). Fractions were centrifuged simultaneously (1600g for 15 min at room temperature) immediately after the spinal puncture and were frozen at –80 °C within 30–40 min. The material was never thawed and refrozen.
We used ABx–40 and ABx–42 methods from The Genetics Co. and Aß1–42, total Tau, and P-tau181P from Innogenetics. Because different antibodies used in the respective assays show different epitope specificities, they enable detection of either specifically one species, namely Aß1–42 [the assay of Innogenetics (11)], or the "family" of Aß peptides ending with a COOH terminus at residue 42 and beginning at different NH2 termini (ABx–42; the assays of The Genetics Co.). All 4 corresponding aliquots from a given patient were applied simultaneously to 1 ELISA plate. All measurements were performed in duplicate. Results are presented in reference to the concentration obtained in PP (set as 100%) and analyzed by ANOVA for repeated measurements, followed by the Scheffé post hoc test.
ABx–40 concentrations were lower in the PS tube than in all other material types (Table 1
; n = 13; P <0.001), whereas concentrations in PC, PX, and PP were similar. CSF ABx–42 concentrations were lower in the PS tube than in all other material tubes (Table 1
; n = 19; P <0.001), whereas there were no significant differences among PC, PX, and PP. The ABx–42/ABx–40 concentration ratio was only slightly higher in PC than in PX (n = 13; P = 0.04), with no differences among other tubes. Aß1–42 was lower on PS (Table 1
) than in PC (P <0.01), PX, and PP (P <0.001). Aß1–42/ABx–42 was higher in PS than in PP and PC (n = 19; P <0.05). Tau was significantly lower (P <0.001) in the PS tube compared with all other tubes. The results also differed between PC and PX (P <0.05; Table 1
). Neither Aß1–42/ABx–40 ratio (n = 13) nor P-tau181P (n = 16) differed significantly among test tubes (Table 1
). Intraassay variability of the biomarkers was similar among the 4 material types with CVs of 1.8%–5.4%). Concentrations of none of the biomarkers differed significantly between the first and the last fraction of the CSF.
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Our study suggests that amyloid ß peptide concentration ratios normalized to the concentration of ABx–40 (i.e., ABx–42/ABx–40 and Aß1–42/ABx–40) and P-tau181P are much less prone to methodologic error introduced by interactions of the biomarkers’ molecules with the test tube surfaces compared with pure Aß peptides concentrations or total tau.
The decreased concentrations of Aß1–42 and total tau in the PS tube agree with the results of Andreasen et al. (4). CSF Aß peptides and tau may adsorb to PS. Because the ABx–42/ABx–40 and Aß1–42/ABx–40 concentration ratios seem not to be affected by the test tube material, we assume that Aß peptides with COOH termini ending at residues 42 and 40 adhere to PS to a similar extent, which might be surprising, as Aß peptides ending at amino acid position 42 (Aß1–42 and ABx–42) are more hydrophobic than is ABx–40 (2)(3). The increase in Aß1–42/ABx–42 in PS compared with PC and PP suggests increased adherence of ABx–42 molecules to PS, which might be explained by the fact that ABx–42 is actually a set of different peptides, beginning at different N-terminal amino acid positions, most probably with different hydrophobic properties. Phosphorylation of the tau molecule increases its hydrophobicity and, presumably, decreases its adsorption to nonpolar plastic.
We suggest that amyloid ß peptide concentration ratios are more reliable biomarkers than are the pure Aß peptides concentrations, as Aß peptides ratios are not altered by interaction with the surface of the collection tubes.
Acknowledgments
This study was supported by the following grants from the German Federal Ministry of Education and Research (BMBF): Kompetenznetz Demenzen (01 GI 0420) and HBPP-NGFN2 (01 GR 0447). We gratefully appreciate the technical help of Christine Schödel.
References
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