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Technical Briefs |
1 Department of Biochemistry,
2 University Department of Surgery, and
3 University Department of Anaesthesia, Royal Infirmary, Glasgow G31 2ER, United Kingdom
aauthor for correspondence: fax 44-0141-553-1703, e-mail dtalwar{at}gri-biochem.org.uk
Vitamin B6 (active 3 hydroxy-2-methylpyridine derivatives) is an essential precursor of pyridoxal (PL) and pyridoxamine phosphate coenzymes of a wide variety of enzymes of intermediary metabolism (1). In plasma, pyridoxal 5'-phosphate (PLP) is the major form, whereas PLP and pyridoxamine 5'-phosphate (PMP) predominate in the cell. The most widely used method to detect vitamin B6 deficiency is the erythrocyte aspartate aminotransferase activation assay (2)(3)(4)(5). As this test is a functional rather than a direct measurement of PLP status, it may be affected by factors other than PLP deficiency (2)(3). The plasma PLP concentration is considered one of the better indicators of vitamin B6 status (1)(6)(7)(8)(9) and is reported to be well correlated with tissue PLP concentrations (8). However, vitamin concentrations in blood cells tend to be a better marker of cellular stores (10). We describe a simple, robust, reversed-phase HPLC method with precolumn derivatization using semicarbazide (11)(12)(13) that is suitable for the simultaneous measurement of PLP, 4-pyridoxic acid (PA), and PL in plasma and red cells and application of the assay in healthy individuals, patients with chronic disease, and critically ill patients requiring intensive care.
Venous blood samples (EDTA) and packed red cells for population reference values were obtained from apparently healthy individuals (laboratory staff and those attending a cardiovascular risk clinic). Samples were obtained from a group of chronically ill medical and surgical patients with the potential for vitamin deficiencies (60% with short bowel syndrome and 35% with either chronic liver or renal disease) and from a critically ill group of medical and surgical patients admitted to the intensive care unit (30% with pancreatitis, 30% with trauma, 30% with organ failure). The study was approved by the local ethics committee of Glasgow Royal Infirmary, and all patients gave informed consent.
PLP and PL in blood were measured as their semicarbazone derivatives, pyridoxal 5'-phosphate semicarbazone (PLPSC) and pyridoxal semicarbazone (PLSC), respectively (12)(13). The assay was optimized with respect to derivatization, separation, and detection. Derivatization was carried out as follows: 500 µL of plasma, calibrator, quality controls, or diluted hemolysate (300 µL of red cells + 700 µL of water) and 40 µL of derivatizing agent (containing 250 g/L semicarbazide and glycine) were combined, vortex-mixed, and left in the dark at room temperature for 30 min. The mixtures were then deproteinized with 40 µL of 700 g/L perchloric acid, vortex-mixed for 1 min, and centrifuged for 10 min (1000g). Each supernatant (300 µL) was stabilized by the addition of 30 µL of 250 g/L NaOH (final pH between 3.0 and 5.0), and 50 µL was injected on the HPLC column via an autosampler (Waters).
HPLC separation was carried out using a Luna C18 reversed-phase column [250 x 4.6 mm (i.d.); 5-µm bead size; protected with a 3 x 4 mm guard column] and an isocratic filtered mobile phase consisting of 60 mmol/L disodium hydrogen phosphate containing 95 mL/L methanol and 400 mg/L EDTA (disodium salt), adjusted to pH 6.8 with concentrated phosphoric acid. The flow rate was 1.5 mL/min. PLPSC and PLSC were detected using a programmable fluorescence detector (Waters). Quantification was by external standardization with a single-concentration plasma calibrator (Chromsystems), using peak heights. Red cell PLP, PA, and PL concentrations were related to hemoglobin (pmol/g Hb). There was no significant loss of PLP, PA, or PL at room temperature from derivatized plasma or red cells over 48 h. The performance of the method was monitored by taking part in the External Quality Assurance scheme for B vitamin (Dusseldorf, Germany).
Albumin and C-reactive protein concentrations in plasma were measured on an automated analyzer (ADVIA 1650; Bayer).
Data from the reference and chronically and critically ill groups are presented as median and range, and ANOVA (Kruskal–Wallis) was carried out. Correlations were carried out using the Spearman rank correlation (SPSS Inc.).
The chromatographic profiles corresponding to a derivatized plasma and a red cell hemolysate extract are shown in Fig. 1
. The PLPSC, PA, and PLSC peaks were well resolved with K' values of 4.7, 11.6, and 19.8, respectively. The peaks were identified by comparing peak retention times with those of PLP, PA, and PL aqueous calibrators after derivatization. For recovery studies, aliquots of a pooled plasma and red cell sample were analyzed both before and after the addition of 40 and 80 nmol/L of PLP, PA, and PL. The recoveries were >90% for all analytes. The within-batch imprecision (CV; n = 10) for PLP, PA, and PL in plasma and red cells was <5%, 12%, and 5%, respectively, from same-day analyses of quality-control material. The between-batch imprecision (n = 17) for PLP, PA, and PL in plasma and red cells was <7%, 14%, and 8%, respectively, from analysis of an aliquoted pooled red cell sample (stored at -70 °C) analyzed over 3 months. The method was linear up to at least 1000 nmol/L for PLP, PA, and PL. The limits of detection, defined as three times the baseline noise, were 2.1 nmol/L for PLP, 1.0 nmol/L for PA, and 2.8 nmol/L for PL. The limits of quantification, defined as 10 times the signal-to-noise ratio, were 5.8 nmol/L for PLP, 2.5 nmol/L for PA, and 6.5 nmol/L for PL.
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Previous methods have used pre- or postcolumn derivatization of PLP to its fluorescent derivative with use of semicarbazide, cyanide, or bisulfite as derivatization agents (2)(14)(15)(16)(17)(18)(19)(20). The major disadvantage of postcolumn derivatization procedures is that because of the photosensitivity of PLP, significant losses of PLP may occur during sample processing and analysis unless these steps are carried out away from ultraviolet light (14)(20). In contrast, we found the precolumn semicarbazide derivatization simple to perform, reliable, and with adequate sensitivity and precision for the routine measurement of PLP in plasma and red cells; it compared favorably with the methods reported by Vuilleumier et al. (2) and Millart et al. (15).
We measured PLP, PA, and PL concentrations in plasma and red cells in healthy individuals and patients with chronic and critical illness (Table 1
). In healthy individuals, the geometric mean (95% reference interval) for PLP was 56 (21–138) nmol/L in plasma and 410 (250–680) pmol/g Hb in red cells, and plasma PLP concentrations correlated with PA (r2 = 0.31; P <0.0001) and PL (r2 = 0.49; P <0.0001). However, in red cells PLP concentrations correlated with PL (r2 = 0.55; P <0.0001) but not with PA and was strongly positively correlated with PLP concentrations in the plasma of healthy individuals (r2 = 0.810; P <0.0001).
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PLP concentrations in the chronically ill group (Table 1
) tended to be lower in plasma (P = 0.03) and red cells (P = 0.05) than in the reference population, but they remained significantly correlated (r2 = 0.412; P <0.0001), and assessment of vitamin B6 status (plasma and red cell PLP) agreed in 28 of the 29 patients. In contrast, in the critically ill group with evidence of systemic inflammatory response and hypoalbuminemia (Table 1
), plasma PLP was significantly lower (P <0.001) and erythrocyte PLP was higher (P = 0.023) than in the reference population. Moreover, plasma and erythrocyte PLP concentrations were only weakly correlated (r2 = 0.358; P = 0.009), and plasma PLP concentrations were below the reference interval in 12 of the 18 patients. However, the majority of the critically ill patients had erythrocyte PLP concentrations within the reference interval.
The reference intervals for PLP, PA, and PL in plasma were in good agreement with data reported by others (15)(16)(17)(18). PLP concentrations in red cells were adjusted to Hb rather than to volume of packed red cells because accurate pipetting of packed red cells is difficult and affects precision. The strong correlation between plasma and red cell PLP concentrations in the reference population suggests that the latter are valid indicators of vitamin B6 status and that either plasma or red cell PLP measurements can be used as markers of vitamin B6 status. This is consistent with the concordance between plasma and red cell PLP concentrations in the chronically ill group. In contrast, in the critically ill group there was little concordance between plasma and red cell PLP concentrations. A possible explanation for these results is that the abnormally low plasma PLP concentrations are related to recent poor nutritional intake, whereas the red cell PLP concentrations reflect vitamin B6 status at the time of red blood cell synthesis. An alternative explanation is redistribution of plasma PLP (the majority of which is bound to albumin) during a pronounced systemic inflammatory response (21). Indeed, the plasma PLP results obtained in the present study are consistent with those reported previously after elective orthopedic surgery (21). Moreover, given that blood samples in this group were taken within 5 days of admission to the intensive care unit and that it is routine practice to give vitamin B supplementation as soon as practicable, the lack of concordance between plasma and red cell PLP concentrations in these patients is likely to be attributable to the low plasma concentrations. However, the present study does not provide a definitive explanation of the results obtained in the critically ill group.
From the results of the present study we conclude that measurement of plasma PLP for assessing vitamin B6 status in patients with a systemic inflammatory response (as evidenced by an increased C-reactive protein concentration) may be misleading. In contrast, red cell PLP concentrations do not decrease in the presence of a systemic inflammatory response and therefore may be useful in differentiating true from apparent vitamin B6 deficiency in patients with a systemic inflammatory response.
References
The following articles in journals at HighWire Press have cited this article:
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  A. T Vasilaki, D. C McMillan, J. Kinsella, A. Duncan, D. S. J O'Reilly, and D. Talwar Relation between pyridoxal and pyridoxal phosphate concentrations in plasma, red cells, and white cells in patients with critical illness Am. J. Clinical Nutrition, July 1, 2008; 88(1): 140 - 146. [Abstract] [Full Text] [PDF]
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J. Dierkes, C. Weikert, K. Klipstein-Grobusch, S. Westphal, C. Luley, M. Mohlig, J. Spranger, and H. Boeing Plasma pyridoxal-5-phosphate and future risk of myocardial infarction in the European Prospective Investigation into Cancer and Nutrition Potsdam cohort Am. J. Clinical Nutrition, July 1, 2007; 86(1): 214 - 220. [Abstract] [Full Text] [PDF]
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 D. K. Talwar, M. K. Azharuddin, C. Williamson, Y. P. Teoh, D. C. McMillan, and D. St. J. O'Reilly Biological Variation of Vitamins in Blood of Healthy Individuals Clin. Chem., November 1, 2005; 51(11): 2145 - 2150. [Abstract] [Full Text] [PDF]
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J. Dierkes, K. Hoffmann, K. Klipstein-Grobusch, C. Weikert, H. Boeing, B.-C. Zyriax, E. Windler, and J. Kratzsch Low plasma pyridoxal-5'phosphate and cardiovascular disease risk in women: results from the Coronary Risk Factors for Atherosclerosis in Women Study Am. J. Clinical Nutrition, March 1, 2005; 81(3): 725 - 727. [Full Text] [PDF]
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