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Letters to the Editor |
1 Department of Clinical Chemistry, and Laboratory Medicine, Faculty of Medicine, University of Saarland, Homburg, Germany
2 Department of Geriatric Rehabilitation, St. Ingbert Hospital, St. Ingbert, Germany
aAddress correspondence to this author at: Department of Clinical Chemistry and Laboratory Medicine, Central Laboratory, University Hospital of the Saarland, Bldg. 57, D-66421 Homburg/Saar, Germany. Fax 49 68411630703; e-mail kchwher{at}uniklinikum-saarland.de.
To the Editor:
After blood collection, homocysteine (Hcy) is generated in blood cells and is continuously released into the plasma. Stabilizing Hcy in blood samples requires immediate sample centrifugation and plasma separation from blood cells, sample cooling, or the use of special collection tubes(1)(2).
To investigate the stability of Hcy and its precursors S-adenosyl-Hcy (SAH) and S-adenosyl-methionine (SAM), we collected fasting blood samples from healthy individuals (n = 8, age 25–46 years) and renal patients (n = 9, GFR 11–48 mL/min, 78–90 years). Patients were recruited from the Department of Internal Medicine IV-Nephrology and Hypertension of the Saarland University Hospital. Controls were selected among hospital employees. The local ethics committee approved the usage of blood samples from patients, and all participants gave informed consent. Samples were collected into EDTA, acidic citrate, and PrimavetteTM tubes. The samples were incubated at 4 °C or at room temperature for 0, 2, 6, and 24 h and were centrifuged afterward. A total of 357 samples were analyzed for Hcy by HPLC (Immundiagnostik) and by fluorescence polarization immunoassay (FPIA; Abbott). Plasma SAM and SAH were determined by a modified liquid chromatography–tandem mass spectrometry method according to Gellekink et al.(3) in 210 samples from a representative subgroup with 10 individuals.
Baseline Hcy values (EDTA) ranged from 5 to 16 µmol/L in healthy individuals and from 9 to 65 µmol/L in renal patients. Baseline plasma SAM and SAH (EDTA) were determined from 68 to 142 and 9 to 16 nmol/L in healthy individuals and from 148 to 392 and 31 to 361 nmol/L in renal patients. The individual increase of Hcy in EDTA samples at room temperature after 24 h was significantly higher (P <0.001) in healthy individuals (8.7–23.6 µmol/L) than in renal patients (0.1–9.5 µmol/L).
Fig. 1
presents the geometric means of plasma Hcy obtained by HPLC and FPIA and plasma SAM and SAH after blood sample incubation over a period of 0, 2, 6, and 24 h at room temperature or at 4 °C. In the Primavette, Hcy obtained by HPLC decreased only slightly (–4.8%) after 6-h incubation at room temperature and returned to the baseline value after 24 h. At 4 °C a significant decrease of Hcy (6.9%) after 24-h incubation was found (P = 0.003). Applying FPIA for the determination of Hcy, no significant changes of Hcy compared with the baseline values were found. However, the comparison between baseline Hcy determined by FPIA and HPLC revealed that Hcy values obtained by FPIA were significantly higher (median difference 11%) than values obtained by HPLC (P <0.001). In EDTA and acidic citrate plasma we observed no significant difference between Hcy obtained by FPIA and HPLC. The positive bias seen with Primavette tubes was likely due to interferences with the FPIA method by its proprietary components, which are kept secret by the manufacturer.
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In acidic citrate samples Hcy increased slowly at room temperature, reaching the level of significance after 6 h (FPIA) and 24 h (HPLC), respectively. At 4 °C Hcy was stable over a 24-h period. In EDTA tubes a strong increase of Hcy was observed that was markedly decreased at 4 °C. In the Primavette, SAM was stable at room temperature and at 4 °C. At room temperature SAM decreased in EDTA and to a smaller extent in acidic citrate tubes. This decrease was clearly decelerated at 4 °C. At room temperature SAH increased in all 3 collection tubes and reached the highest values in the Primavette after 24 h. The increase was attenuated at 4 °C.
In Primavette and acidic citrate samples we observed 7- and 3-fold increases of plasma SAH after 24-h incubation at room temperature, whereas Hcy was stable and increased by 20%, respectively (geometric means). In EDTA samples SAH and Hcy increased 1.8-fold. Interestingly, the increase of SAH after 24 h correlated negatively with the increase of Hcy after 24 h in Primavette and acidic citrate samples (r = –0.647; P = 0.002) but not in EDTA samples (r = 0.067). Hcy is generated in erythrocytes from its precursor SAH by catalysis of SAH hydrolase. Therefore, the inhibition of SAH hydrolase activity causes an increase of SAH and no or low increase of Hcy. In contrast, in EDTA samples the increase of SAH is low because SAH is metabolized to Hcy. Furthermore, a leakage of SAH from erythrocytes into the plasma might occur because cellular SAH concentrations are approximately 10-fold higher than in plasma(4). In blood samples Hcy can be stabilized by the addition of an SAH hydrolase inhibitor(5).
In conclusion, our results indicate that the stabilizing effect of Primavette and acidic citrate on Hcy is due to the inhibition of SAH hydrolase activity. The inhibition of SAH hydrolase is more efficient in Primavette than in acidic citrate tubes. However, in Primavette samples Hcy obtained by FPIA was approximately 11% higher than Hcy obtained by HPLC.
Acknowledgments
Grant/funding support: None declared.
Financial disclosures: None declared.
References
The following articles in journals at HighWire Press have cited this article:
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  T. Tamura and J. E. Baggott In Vitro Formation of Homocysteine in Whole Blood in the Presence of Anticoagulants Clin. Chem., August 1, 2008; 54(8): 1402 - 1403. [Full Text] [PDF]
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, acidic citrate; 
, Primavette).