Clin Chem Lab Med 2014; 52(9): e203–e204
Letter to the Editor Antoine Huguenin, Jean-Baptiste Oudart, Julie Hubert, François-Xavier Maquart and Laurent Ramont*
Serum folate and vitamin B12: does light really matter? Keywords: folate; light; vitamin B12. DOI 10.1515/cclm-2014-0254 Received March 7, 2014; accepted April 6, 2014; previously published online May 1, 2014
To the Editor, Vitamin B12 (cobalamin) and folate (vitamin B9) are enzyme cofactors essential for important metabolic pathways such as DNA synthesis. The assay of these vitamins is of major interest in the diagnosis of megaloblastic anemia [1] and in the evaluation of some neurological disorders (subacute combined degeneration of the spinal cord and peripheral neuropathy). Vitamin B12 and folate have long been known to be photolabile when exposed to ultraviolet radiation. This photodegradation is influenced by endogenous photosensitizers [2] (riboflavin, bilirubin or uroporphyrin) or photoprotectors, such as serum albumin [3], present in biological samples. According to the manufacturer’s instructions for the assays, folate and vitamin B12 samples should be protected from light. A major consequence of such recommendation is that it most often prevents the use of pre-analytical automated systems, which are commonly used in most laboratories, for pre-sorting, centrifugation and aliquoting, and makes it necessary to process these samples manually. However, an analysis of the literature shows that this recommendation is supported by very few data obtained in daily practice. This prompted us to investigate if light exposition in common laboratory *Corresponding author: Dr. Laurent Ramont, Laboratoire Central de Biochimie, CHU de Reims, Rue Serge Kochman 51092 Reims Cedex, France, Phone: +33 326783181, Fax: +33 326788539, E-mail: [email protected]; and CNRS UMR 7369, Université de Reims-Champagne-Ardenne, Reims, France Antoine Huguenin and Julie Hubert: CHU de Reims, Laboratoire Central de Biochimie, Reims, France Jean-Baptiste Oudart and François-Xavier Maquart: CHU de Reims, Laboratoire Central de Biochimie, Reims, France; and CNRS UMR 7369, Université de Reims-Champagne-Ardenne, Reims, France
settings actually causes significant serum vitamin B12 and folate decay. This study was based on samples collected after medical prescription of vitamin B12 and/or folate assay in the Reims University Hospital (Reims, France). Blood was collected in Vacutainer® tubes (BD™, Le Pont de Claix, France) with coagulation activator. Specimens were protected from light immediately after blood sampling by wrapping in tin foil. Samples were centrifuged at 2000 g, for 10 min at 4 °C. Serums were assayed immediately (t0h) then left unprotected under ambient light (mixture of artificial and natural light) at room temperature, and reassayed after 2 h (t2h) and 4 h (t4h) of light exposition. All tests were performed using electrochemiluminescent immunoassay (Roche Elecsys Vitamin B12® and Elecsys Folate III® assays) on COBAS 8000 automated analyzer (Roche™, Meylan, France). Differences between groups were analyzed by ANOVA and Pearson’s test, the degree of agreement between measurements was evaluated using the Bland-Altman Analysis with Microsoft Excel 2007 StatEL® (@Science). Serum folate was assayed on 104 samples. Mean level was 6.31 ng/mL (max–min: 1.62–18.17 ng/mL, median 5.82, standard deviation: 2.93). At t0h, 31 (30%) patients were considered as folate deficient ( 18.6 ng/mL). There was no significant difference in the folate level (Figure 1A) between t0h, t2h and t4h (p = 0.78; ANOVA). Among the 31 patients who were classified as deficient at t0h, only two were classified as normal at 4 h. Inversely, among the 73 patients classified as normal at t0h, only six were classified as deficient at t4h. In every case, the values were very close to the cut-off value and all the variations remained included in the inaccuracy limits of the technique. Correlation using Spearman gave coefficient R2 = 0.957 between t0h and t2h and R2 = 0.923 between t0h and t4h. Except for one discordant value, explained by a technical problem, all the variations of the results remained included in the inaccuracy limits of the technique.
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e204 Huguenin et al.: Vitamins (B9 and B12) and light
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4
B
200 1.867
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-2
-50
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-100
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Mean folate levels, ng/mL
-69.38 0
500 1000 1500 Mean vitamin B12 levels, pg/mL
Figure 1 Bland and Altman plot of folate (A) or vitamin B12 (B) measurements at t0h compared to t4h. Abscissa shows the mean level of each sample at t0h and t4h. Ordered show the difference between the level measured at t0h and at t4h. Full line represent mean of all differences and dotted lines represent 1.95 ± standard deviation. Cross shows discordant value.
Serum vitamin B12 was assayed on 103 samples. Mean level was 564 pg/mL (min–max: 129–1706, median: 436, standard deviation: 351.96). At t0h, three patients (3%) were considered as vitamin B12 deficient ( 663 pg/mL). There was no significant difference in the serum vitamin B12 level (Figure 1B) between t0h, t2h and t4h (p = 0.81; ANOVA). Only one patient considered as normal ( > 191 pg/mL) at t0h was classified as deficient at t4h. Correlation using Spearman’s coefficient were R2 = 0.989 between t0h and t2h and R2 = 0.984 between t0h and t4h. All the variation of the results remained included in the inaccuracy limits of the technique. Vitamin B12 and folate assays are widely practiced but only a few studies have been published on the effect of light exposure on serum folate and vitamin B12 in routine analytical conditions. Moreover, these reports included a low number of samples (n = 9) [4], or healthy volunteers (n = 18) [5]. Our results plead for a low impact of ambient light on folate and vitamin B12 assays in serum samples in routine condition. We observed few discrepancies (differences > 1.96 SD after 4 h) at high values ( > 10 ng/mL for folate and > 800 pg/mL for vitamin B12) with no consequence for clinical interpretation of the results. Samples for analysis of vitamin B12 and folate are traditionally processed manually for photoprotection. This study demonstrates the high stability of these analytes in standard laboratory conditions, even without photoprotection, and provided that assay is performed less than 4 h after the arrival of the sample in the laboratory. A 4 h period is a reasonable time for one site (i.e., hospital) laboratories.
The current results are only reliable in the time frame evaluated. Photoprotection remains recommended when a longer period of time is necessary for transportation of the sample to the laboratory. Suppressing photoprotection in the laboratory allows the use of pre-analytical automated system for pre-sorting, centrifugation and aliquoting, which greatly simplifies the pre-analytical management of vitamin B12 and folate assay samples. Conflict of interest statement Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared.
References 1. Wickramasinghe SN. Diagnosis of megaloblastic anaemias. Blood Rev 2006;20:299–318. 2. Juzeniene A, Tam TT, Iani V, Moan J. 5-Methyltetrahydrofolate can be photodegraded by endogenous photosensitizers. Free Radic Biol Med 2009;47:1199–204. 3. Vorobey P, Steindal AE, Off MK, Vorobey A, Moan J. Influence of human serum albumin on photodegradation of folic acid in solution. Photochem Photobiol 2006;82:817. 4. Mastropaolo W, Wilson MA. Effect of light on serum B12 and folate stability. Clin Chem 1993;39:913. 5. Clement NF, Kendall BS. Effect of light on vitamin B12 and folate. Lab Med 2009;40:657–9.
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Letter to the Editor Antoine Huguenin, Jean-Baptiste Oudart, Julie Hubert, François-Xavier Maquart and Laurent Ramont*
Serum folate and vitamin B12: does light really matter? Keywords: folate; light; vitamin B12. DOI 10.1515/cclm-2014-0254 Received March 7, 2014; accepted April 6, 2014; previously published online May 1, 2014
To the Editor, Vitamin B12 (cobalamin) and folate (vitamin B9) are enzyme cofactors essential for important metabolic pathways such as DNA synthesis. The assay of these vitamins is of major interest in the diagnosis of megaloblastic anemia [1] and in the evaluation of some neurological disorders (subacute combined degeneration of the spinal cord and peripheral neuropathy). Vitamin B12 and folate have long been known to be photolabile when exposed to ultraviolet radiation. This photodegradation is influenced by endogenous photosensitizers [2] (riboflavin, bilirubin or uroporphyrin) or photoprotectors, such as serum albumin [3], present in biological samples. According to the manufacturer’s instructions for the assays, folate and vitamin B12 samples should be protected from light. A major consequence of such recommendation is that it most often prevents the use of pre-analytical automated systems, which are commonly used in most laboratories, for pre-sorting, centrifugation and aliquoting, and makes it necessary to process these samples manually. However, an analysis of the literature shows that this recommendation is supported by very few data obtained in daily practice. This prompted us to investigate if light exposition in common laboratory *Corresponding author: Dr. Laurent Ramont, Laboratoire Central de Biochimie, CHU de Reims, Rue Serge Kochman 51092 Reims Cedex, France, Phone: +33 326783181, Fax: +33 326788539, E-mail: [email protected]; and CNRS UMR 7369, Université de Reims-Champagne-Ardenne, Reims, France Antoine Huguenin and Julie Hubert: CHU de Reims, Laboratoire Central de Biochimie, Reims, France Jean-Baptiste Oudart and François-Xavier Maquart: CHU de Reims, Laboratoire Central de Biochimie, Reims, France; and CNRS UMR 7369, Université de Reims-Champagne-Ardenne, Reims, France
settings actually causes significant serum vitamin B12 and folate decay. This study was based on samples collected after medical prescription of vitamin B12 and/or folate assay in the Reims University Hospital (Reims, France). Blood was collected in Vacutainer® tubes (BD™, Le Pont de Claix, France) with coagulation activator. Specimens were protected from light immediately after blood sampling by wrapping in tin foil. Samples were centrifuged at 2000 g, for 10 min at 4 °C. Serums were assayed immediately (t0h) then left unprotected under ambient light (mixture of artificial and natural light) at room temperature, and reassayed after 2 h (t2h) and 4 h (t4h) of light exposition. All tests were performed using electrochemiluminescent immunoassay (Roche Elecsys Vitamin B12® and Elecsys Folate III® assays) on COBAS 8000 automated analyzer (Roche™, Meylan, France). Differences between groups were analyzed by ANOVA and Pearson’s test, the degree of agreement between measurements was evaluated using the Bland-Altman Analysis with Microsoft Excel 2007 StatEL® (@Science). Serum folate was assayed on 104 samples. Mean level was 6.31 ng/mL (max–min: 1.62–18.17 ng/mL, median 5.82, standard deviation: 2.93). At t0h, 31 (30%) patients were considered as folate deficient ( 18.6 ng/mL). There was no significant difference in the folate level (Figure 1A) between t0h, t2h and t4h (p = 0.78; ANOVA). Among the 31 patients who were classified as deficient at t0h, only two were classified as normal at 4 h. Inversely, among the 73 patients classified as normal at t0h, only six were classified as deficient at t4h. In every case, the values were very close to the cut-off value and all the variations remained included in the inaccuracy limits of the technique. Correlation using Spearman gave coefficient R2 = 0.957 between t0h and t2h and R2 = 0.923 between t0h and t4h. Except for one discordant value, explained by a technical problem, all the variations of the results remained included in the inaccuracy limits of the technique.
Brought to you by | Karolinska Institute Authenticated Download Date | 5/22/15 10:35 PM
e204 Huguenin et al.: Vitamins (B9 and B12) and light
A
4
B
200 1.867
2 1 0 -1
-1.32
Differences t0h-t4h
Differences t0h-th4
3
250
150 128.7 100 50 0
-2
-50
-3
-100
0
5
10
15
20
Mean folate levels, ng/mL
-69.38 0
500 1000 1500 Mean vitamin B12 levels, pg/mL
Figure 1 Bland and Altman plot of folate (A) or vitamin B12 (B) measurements at t0h compared to t4h. Abscissa shows the mean level of each sample at t0h and t4h. Ordered show the difference between the level measured at t0h and at t4h. Full line represent mean of all differences and dotted lines represent 1.95 ± standard deviation. Cross shows discordant value.
Serum vitamin B12 was assayed on 103 samples. Mean level was 564 pg/mL (min–max: 129–1706, median: 436, standard deviation: 351.96). At t0h, three patients (3%) were considered as vitamin B12 deficient ( 663 pg/mL). There was no significant difference in the serum vitamin B12 level (Figure 1B) between t0h, t2h and t4h (p = 0.81; ANOVA). Only one patient considered as normal ( > 191 pg/mL) at t0h was classified as deficient at t4h. Correlation using Spearman’s coefficient were R2 = 0.989 between t0h and t2h and R2 = 0.984 between t0h and t4h. All the variation of the results remained included in the inaccuracy limits of the technique. Vitamin B12 and folate assays are widely practiced but only a few studies have been published on the effect of light exposure on serum folate and vitamin B12 in routine analytical conditions. Moreover, these reports included a low number of samples (n = 9) [4], or healthy volunteers (n = 18) [5]. Our results plead for a low impact of ambient light on folate and vitamin B12 assays in serum samples in routine condition. We observed few discrepancies (differences > 1.96 SD after 4 h) at high values ( > 10 ng/mL for folate and > 800 pg/mL for vitamin B12) with no consequence for clinical interpretation of the results. Samples for analysis of vitamin B12 and folate are traditionally processed manually for photoprotection. This study demonstrates the high stability of these analytes in standard laboratory conditions, even without photoprotection, and provided that assay is performed less than 4 h after the arrival of the sample in the laboratory. A 4 h period is a reasonable time for one site (i.e., hospital) laboratories.
The current results are only reliable in the time frame evaluated. Photoprotection remains recommended when a longer period of time is necessary for transportation of the sample to the laboratory. Suppressing photoprotection in the laboratory allows the use of pre-analytical automated system for pre-sorting, centrifugation and aliquoting, which greatly simplifies the pre-analytical management of vitamin B12 and folate assay samples. Conflict of interest statement Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared.
References 1. Wickramasinghe SN. Diagnosis of megaloblastic anaemias. Blood Rev 2006;20:299–318. 2. Juzeniene A, Tam TT, Iani V, Moan J. 5-Methyltetrahydrofolate can be photodegraded by endogenous photosensitizers. Free Radic Biol Med 2009;47:1199–204. 3. Vorobey P, Steindal AE, Off MK, Vorobey A, Moan J. Influence of human serum albumin on photodegradation of folic acid in solution. Photochem Photobiol 2006;82:817. 4. Mastropaolo W, Wilson MA. Effect of light on serum B12 and folate stability. Clin Chem 1993;39:913. 5. Clement NF, Kendall BS. Effect of light on vitamin B12 and folate. Lab Med 2009;40:657–9.
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