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Relation between pyridoxal and pyridoxal phosphate concentrations in plasma, red cells, and white cells in patients with critical illness^1,2,3

¹ From the University Departments of Surgery (ATV and DCM) and Anaesthesia (DTV and JK) and the Department of Biochemistry (ATV, AD, DSJO, and DT), Royal Infirmary, Glasgow, United Kingdom

² Supported by the University Departments of Surgery, Anaesthesia, and Biochemistry, Royal Infirmary, Glasgow, United Kingdom.

³ Reprints not available. Address correspondence to DC McMillan, University Department of Surgery, Royal Infirmary, Glasgow G31 2ER, United Kingdom. E-mail: d.c.mcmillan{at}clinmed.gla.ac.uk.

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Background:Evidence suggests that the relation between plasma and red cell vitamin B-6 concentrations is perturbed as part of the systemic inflammatory response in critically ill patients.

Objective:The aim was to examine the cross-sectional and longitudinal interrelations between pyridoxal (PL) and pyridoxal phosphate (PLP) concentrations in plasma and red and white cells in patients with critical illness.

Design:PLP and PL concentrations were measured by HPLC in plasma and red and white cells in normal subjects (n = 126) and critically ill patients (n = 96) on admission and on follow-up.

Results:On admission, compared with the controls, median plasma PLP and PL (P < 0.001 and < 0.01, respectively) and red cell PLP and PL (P < 0.001 and < 0.05, respectively) and their ratio (PLP:PL) in plasma and red cells (P < 0.001 and < 0.01, respectively) were significantly lower in the critically ill. In critically ill patients, plasma PLP:PL was significantly lower than red cell PLP:PL (P = 0.001) and white cell PLP:PL (P = 0.008). Plasma PL concentration was directly associated with both red cell PL (r_s = 0.73, P < 0.001) and white cell PL (r_s = 0.68, P < 0.001). Red cell PL and white cell PL were directly associated with red cell PLP (r_s = 0.82, P < 0.001) and white cell PLP (r_s = 0.68, P < 0.001), respectively. Longitudinal measurements (n = 48) were similar.

Conclusions:The relation between plasma PLP and PL was significantly perturbed in critical illness. This effect was less pronounced in red and white cells. Therefore, these results confirm the hypothesis that intracellular PLP concentrations are more likely to be a reliable measure of status than are plasma measurements in the critically ill patient.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Vitamin B-6 is an essential precursor of pyridoxal (PL) and pyridoxamine phosphate coenzymes of a variety of enzymes of intermediary metabolism (1). It is recognized that the active form of vitamin B-6 in plasma and tissue is pyridoxal phosphate (PLP). In health, plasma PLP appears to be determined primarily by the intake of vitamin B-6, its binding to albumin, and its hydrolysis to PL by alkaline phosphatase (2, 3).

In contrast to functional tests, direct measurement of PLP concentrations in plasma is thought to most accurately reflect nutritional status of vitamin B-6 (4, 5). In health, PLP concentrations in plasma and tissues are determined mainly by the intake and conversion of vitamin B-6 to PLP and then to PL; therefore, there is a strong association in the plasma and in the tissues between PLP and PL (6, 7). However, recent work has shown that, as part of the systemic inflammatory response, plasma PLP concentrations are reduced such that the relation between plasma and red cell PLP concentrations is disturbed (8, 9). For example, in patients with critical illness, supplementation with vitamin B-6 is associated with an increase in concentrations of PLP in the red cell but not in the plasma (10). The mechanisms underlying this observation are as yet unclear. Cheng et al (11) reported that, during vitamin B-6 supplementation in critically ill patients, plasma PL concentrations increased 15–20-fold, whereas plasma PLP concentrations only increased 3-fold. However, it is unclear whether the reduced plasma PLP concentrations in critically ill patients are due to reduced binding of PLP to albumin, increased hydrolysis of PLP by alkaline phosphatase, or both. Moreover, to our knowledge, the relation between the intracellular PL and PLP concentrations has not been previously examined in the critically ill patient.

Therefore, the aim of the present study was to examine the cross-sectional and longitudinal interrelations between PL and PLP concentrations in plasma, red cells, and white cells in patients with critical illness. This information is required to assess the reliability of these measurements as indicators of vitamin B-6 status in the critically ill patient.

	SUBECTS AND METHODS

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Controls, patients, and study design
Blood samples for population reference values were obtained from laboratory staff, from local health centers, and from people attending a cardiovascular risk clinic. None of the subjects were taking any vitamin supplements or had any significant medical history or evidence of a systemic inflammatory response (serum C-reactive protein <10 mg/L). No formal diet histories were taken (7). Patients in the intensive therapy unit (ITU) of the Royal Infirmary, Glasgow, who had respiratory failure that required ventilatory support, were 18 y old, and who had evidence of the systemic inflammatory response syndrome as per the criteria of Bone et al (12), were studied.

Venous blood samples (EDTA) were withdrawn on admission (day 1) and a further sample between days 2 and 12 for the analysis of plasma, red cell, and white cell vitamin B-6. Blood concentrations of C-reactive protein, albumin, hemoglobin, and alkaline phosphatase were also measured. Acute Physiology and Chronic Health Evaluation II (APACHE II)–predicted mortality and vitamin B complex supplementation were recorded. Enteral feeding was usually instituted on the second day in the ITU and provided only recommended dietary allowance (1.3 mg/d) concentrations of vitamin B-6.

Patients received vitamin B complex supplementation if they were considered malnourished or had a history of excessive alcohol intake. Vitamin B complex supplementation was given as Pabrinex (Link Pharmaceuticals Ltd, West Sussex, United Kingdom), one dose of which provided 50 mg vitamin B-6 hydrochloride. In those patients who received vitamin B supplementation a single dose of Pabrinex was given on the morning of day 2 and daily during their ITU stay. Some patients received additional doses such that the median number of doses received was 3/d in patients who had follow-up measurements.

With respect to the critically ill patients, the study was approved by the ethics committees of the North Glasgow National Health Service Trust and the Multicenter Research Ethics Committee Scotland. When patients were unable to give signed informed consent, consent was obtained from the patients' next of kin or welfare guardian in accordance with the requirements of the Adults with Incapacity Scotland (2000) Act.

Collection and preparation of blood samples
The EDTA samples were centrifuged (500 x g, 4 °C, 10 min), and plasma was removed into another plastic tube for measurements of plasma PLP and PL. After removing the buffy coat, the remaining packed red cells were kept for red blood cell vitamin B-6 determination. All tubes were stored at –70 °C until analysis. All samples were protected from light and assayed in a single batch for each of the analytes to minimize interbatch analytic variation (7).

White cell preparation
One milliliter of EDTA whole blood was transferred into a 15-mL (16 x 100 mm) conical tube, and the red blood cells were lysed, within 24 h of blood sampling, with 9 mL of cold ammonium chloride (8.3 g/L) containing EDTA (372 mg/L) solution. EDTA was shown to prevent platelet binding to leukocytes (13). To minimize damage to white cells, resulting in low leukocyte yields, the pH of the lysing solution was adjusted to 7.4 (14). The samples were gently mixed for 2 min and kept at –15 °C for 10 min, to avoid the creation of clumps and to simplify the elimination of hemoglobin (14).

The samples were then centrifuged (200 x g, 4 °C, 10 min), and the red supernatant fluid containing hemoglobin was discarded. The remaining pellet containing enriched white cells was washed with 10 mL of cold Dulbecco's phosphate-buffered saline (Sigma Chemical, St Louis, MO). This process was performed twice. The samples were centrifuged again but at 100 x g to keep the lighter platelets in the supernatant fluid so they could be removed. The supernatant fluid was removed, the resulting pellet was dispersed in 1 mL of phosphate-buffered saline, and the number of cells per liter was counted with a hemocytometer (KX–21N; Sysmex UK Ltd, Milton Keynes, United Kingdom). In addition, white blood cells and lymphocyte numbers were measured in whole blood by the same counter. The method provided a white cell extraction yield of 63% (n = 76, 50–93%) containing mainly neutrophils (>90%; A Vasilaki, DC McMillan, J Kinsella, A Duncan, DSJ O'Reilly, and D Talwar, unpublished data, 2007).

After a final brief centrifugation (5000 x g, room temperature, 3 min), the supernatant fluid was removed, and the resulting pellet was dispersed in 250 µL of deionized water. The cell suspension was stored at –80 °C for PL and PLP analysis. Before analysis, the cell suspension was sonicated for 10 min to ensure complete lysis of the cells.

Laboratory measurement of PLP and PL
PLP and PL concentrations were measured in plasma, red cells, and white cells by HPLC with the use of precolumn semicarbazide derivatization and fluorescent detection (7, 8). In brief, plasma (500 µL), diluted red cell hemolysates (300 µL of red cells in 700 µL of water), or white cell preparation (250 µL) were derivatized with semicarbazide. The mixtures were then deproteinized with perchloric acid, stabilized with sodium hydroxide, and injected on the HPLC column by an autosampler (Waters, Watford, United Kingdom) (7).

The within-batch imprecision for plasma PLP was 4.9% at 59 nmol/L and 6.3% at 16 nmol/L, for red cell PLP the within-batch imprecision was 5.2% at 367 pmol/g hemoglobin (7), and for white cell PLP the within-batch imprecision was 3.4% at 1.64 nmol/10⁹ cells (A Vasilaki, DC McMillan, J Kinsella, A Duncan, DSJ O'Reilly, and D Talwar, unpublished data, 2007). The within-batch imprecision for plasma PL was 4.6% at 36 nmol/L and 3.0% at 144 nmol/L, and for red cell PL the within-batch imprecision was 4.6% at 36 pmol/g hemoglobin.

PLP concentrations in red cells were adjusted to hemoglobin rather than to the volume of packed red cells because accurate pipetting of packed red cells is difficult because of high viscosity. The 95% reference intervals for the above assays established in our laboratory were as follows: plasma PLP, 17–135 nmol/L; plasma PL, 5–26 nmol/L; red cell PLP, 250–680 pmol/g hemoglobin, and red cell PL, 25–195 pmol/g hemoglobin.

Albumin, C-reactive protein, and alkaline phosphatase were measured by routine laboratory procedures with the use of an automated analyser (Architect; Abbott Diagnostics, Maidenhead, United Kingdom). The limit of detection for albumin was 10 g/L. The limit of detection for C-reactive protein was 5 mg/L. The limit of detection for alkaline phosphatase was 5.0 U/L. The interassay CV was <5% over the sample concentration range for albumin, C-reactive protein, and alkaline phosphatase.

Hemoglobin estimation was performed with the use of Drabkins Reagent (Sigma Diagnostics, Gillingham, United Kingdom). Hemoglobin is oxidized and converted to stable cyanmethemoglobin, and the absorbance was measured at the main wavelength of 546 nm with the use of automatic analyser (Sapphire 350; Audit Diagnostics, Carrigtwohill, Ireland). The within-batch imprecision CV was 0.95% at 6.9 g/dL. The between-batch imprecision CV was 4.7% at 7.1 g/dL.

Statistics
Data from normal subject and critically ill patient groups are presented as median and range. Comparisons between the control and critically groups were performed with the use of the Mann-Whitney U test. Correlations between variables in the critically ill group were performed with the use of the Spearman's rank correlation. Data from different time points in the patient groups were tested for statistical significance with the use of the Wilcoxon's signed-rank test. Because of the number of statistical comparisons, a P value of < 0.01 was considered to be significant. Analysis was performed with the use of SPSS software (version 15; SPSS Inc, Chicago, IL).

	RESULTS

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The baseline characteristics of controls (n = 126) and critically ill patients (n = 96) studied are shown in Table 1. Most patients were men, >50 y of age, and similar to the control group. The patients' median APACHE II score was 20, and the associated median predicted mortality was 32%. Most patients had low concentrations of hemoglobin and albumin and high concentrations of C-reactive protein and alkaline phosphatase. There were 96 patients admitted to the intensive care unit who had plasma PLP and also PL concentrations measured. Of those patients 74 also had red cell and white cell PLP and PL concentrations measured. Compared with the control group, median plasma PLP, PL, and their ratio (PLP:PL) were significantly lower in the critically ill group (P < 0.001, P < 0.01, and P < 0.001, respectively). Compared with the control group, median red cell PLP and the red cell PLP:PL were significantly lower in the critically ill group (P < 0.001 and P < 0.01, respectively). White blood cell vitamin B-6 concentrations were not measured in the controls because this was not part of the original protocol for establishing reference intervals in a healthy population; therefore, they could not be compared with values in the critically ill group. The correlations between plasma PLP and red cell PLP were 0.90 (P < 0.001) and 0.46 (P < 0.001) in the control and the critically ill group, respectively. In the critically ill patients the plasma PLP:PL was significantly lower than was the red cell PLP:PL (P = 0.001) and white cell PLP:PL (P = 0.008). In contrast, the red cell and white cell PLP:PL were not significantly different (P = 0.515).

Table 2

Table 3

Table 4

Table 5

	DISCUSSION

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Few studies have examined the relation between plasma and intracellular concentrations of PL and PLP. Two such studies have reported relations in small numbers of healthy subjects (16, 17). Hamfelt (16) reported that, in 10 healthy children and 37 adults, there were significant correlations between plasma, red cell, and white cell concentrations of PLP. However, it was noted that the correlation between plasma and white cell PLP concentrations appeared to be weaker than the corresponding plasma and red cell concentrations (16). Also, Shephard et al (17) reviewed the concentrations of PLP in plasma, red cells, and white cells of healthy subjects. In the present study, we did not measure PLP in the white cells of healthy subjects. However, the concentration of PLP in white cells was approximately 2-fold higher in the critically ill patients than with previous concentrations reported in the literature (17).

With respect to comparison of the present red cell PLP concentrations in healthy subjects to previous reports (16, 17), this is problematical because of methodologic differences. For example, both those previous reports used postcolumn derivatization, whereas the present study used precolumn semicarbazide derivatization. It is recognized that the acid precipitation in the postcolumn derivatization method may lead to suboptimal extraction of PLP from red cells (7, 18).

The results of the present study show that, in plasma, red cells, and white cells, PLP is strongly and directly associated with the concentrations of PL in patients with critical illness. It was of interest that the plasma PLP:PL in critically ill patients on admission and on follow-up was much lower than that of the plasma controls. There were also similar findings with the red cell PLP:PL in controls and critically ill patients. However, on admission the reduction of the PLP:PL was greater in the plasma (55%) than in the red cells (18%). To our knowledge, the present study shows, for the first time, that the relation between plasma PLP and PL in controls is similar to that found in red and white cells in patients with critical illness. Given that, compared with plasma PLP, concentrations of plasma PL are more strongly correlated with those in the red or white cells and that red cell and white cell PL concentrations are strongly and similarly correlated with their respective PLP concentrations show the importance of PL in the intracellular metabolism of PLP in normal subjects and critically ill patients. One interpretation of the results of the present study might be to suggest that plasma PL would be a good surrogate measure of intracellular PLP concentrations. However, PL is not the physiologically active form of vitamin B-6; therefore, the clinical relevance of measuring plasma PL concentrations is not certain. However, we believe that the present intracellular measurements of PLP, compared with plasma, are a more accurate reflection of vitamin B-6 status and should be used in routine assessment of the patient with critical illness.

The results of the present study are therefore consistent with previous small cross-sectional studies, which questioned the use of plasma PLP as a marker of vitamin B-6 status in subjects with evidence of a systemic inflammatory response (8, 9). There are parallels between the present study of vitamin B-6 and previous work on selenium and glutathione peroxidase activity (19). Also, it was recently reported that the prognostic value of plasma PLP concentrations, as a marker of myocardial infarction risk, can be accounted for, in large part, by the presence of a systemic inflammatory response as evidenced by an elevated concentration of C-reactive protein (20).

The basis of the relatively low plasma PLP concentrations in these patients is not clear. However, albumin, whose binding appears to protect PLP from hydrolysis, was low both on admission and on follow-up. Furthermore, albumin was directly associated with plasma PLP both in the cross-sectional measurements and the longitudinal changes. Interestingly, Keniston et al (21, 22) reported that the ratio of PLP concentration in deproteinized (bound and unbound PLP) and nondeproteinized (unbound PLP) plasma samples varies with clinical condition. Because albumin is the main binding protein in PLP in plasma, they concluded that albumin concentration affects PLP concentration in plasma. Indeed, because albumin is readily redistributed from plasma, as part of the systemic inflammatory response, this is consistent with the observation that plasma concentrations of vitamin B-6 are transiently decreased in subjects undergoing elective surgery (9) and that vitamin-B6 supplementation in critically ill patients was unable to increase plasma PLP concentration (10, 23, 24).

Alkaline phosphatase is another potentially important determinant of plasma PLP concentrations in patients with critical illness. In contrast to albumin, the alkaline phosphatase activity, although highly elevated on admission and on follow-up, was not associated with plasma PLP either in the cross-sectional or longitudinal studies. However, the laboratory measurement of alkaline phosphatase might not indicate the true functional activity because the analytic method requires dilution of the serum sample, and serum phosphate concentration in the clinically observed range inhibits alkaline phosphatase activity under physiologic conditions (25). Clearly, this may have contributed to the weak correlation observed in the present study between plasma PLP concentrations and measured alkaline phosphatase activity. These results are consistent with the concept that plasma PLP concentrations are determined, at least in part, by its binding to albumin and that any free PLP is subject to hydrolysis to PL by alkaline phosphatase (3).

The results of the present study do not address the question of increased use and metabolic turnover of plasma PLP. However, the results of studies of Cheng et al (11) and Huang et al (26) show that the metabolic end product of PLP, pyridoxic acid, is significantly increased both in the plasma and in the urine of critically ill patients. Taken together with the results of the present study would suggest that the low concentrations of vitamin B-6 in plasma are influenced both by increased redistribution and catabolism.

Patients admitted to the intensive care unit are under severe metabolic stress and may have increased utilization and consumption of vitamin B-6; therefore, some researchers have advocated for the supplementation of vitamin B-6 in these patients, which appears to have a beneficial effect on immune responses (11, 26). In the present study the extreme high concentrations of red cell PL and PLP were measured in those patients who had recorded supplementation. Few patients had no recorded supplementation and therefore precluded meaningful comparison of the supplemented and nonsupplemented groups. Therefore, it is likely that the extreme values reflect the effect of supplementation and not analytic or methodologic error. Indeed, it was of interest that, on admission, a few patients had extremely high concentrations of red cell PLP and PL, suggesting that some patients had received supplementation before admission and would confirm the utility of red cell measurements. Furthermore, it is not clear whether red or white cells should be used, in preference to plasma. However, taking into consideration the higher sensitivity and that red cells are simpler to separate and analyze, we would recommend red cell analysis for assessment of vitamin B-6 status and to guide supplementation in patients with critical illness.

The question of whether red cell concentrations are more likely to detect deficiencies or toxicity in these patients cannot be definitively answered by the present study because intracellular concentrations were not measured in other tissues. With respect to red cell PLP, it was proposed as a more relevant measure of vitamin B-6 status because the site of PLP coenzyme function is intracellular (27, 28). In addition, in health red cell PLP concentrations were shown to be associated with the dietary intake of vitamin B-6, with vitamin B-6 supplementation, and with the functional tests used to assess vitamin B-6 status (29–31). This work showed that red cell values were less sensitive to acute changes than were plasma values. Nevertheless, in future studies it will be important to establish whether deficiencies in red cell concentrations of vitamin B-6 are related to outcome in the critically ill patients.

Although little evidence from the literature suggests that supplementation with vitamin B-6 is toxic, the doses given in the present study (median: 150 mg/d) are above the recommendations which have, on the basis of the development of sensory neuropathy, set the tolerable upper intake value of 100 mg/d (32). Given that there is significant accumulation in the red cells (30 times the upper limit of normal values in some patients) and assuming that red cell PLP concentrations reflect the concentrations in other tissues, there is evidence of significant accumulation in patients with critical illness. It would therefore be reasonable to adopt a cautious approach to vitamin B-6 supplementation in these patients.

Therefore, given the results of the present study, it would be important to monitor red cell PLP concentrations to identify patients with evidence of excessive cellular accumulation of vitamin B-6 and to regulate subsequent vitamin B-6 supplementation accordingly. In the Royal Infirmary, Glasgow, we routinely monitor for accumulation of vitamin B-6 (PLP) in red blood cells in critically ill patients. On the basis of these results we advise that there is significant tissue accumulation of PLP in red cells, when concentrations in red cells are >4000 pmol/g hemoglobin (5 times the upper limit of normal), and the potential risk of toxicity.

In summary, the relation between plasma PLP and PL is disturbed in patients with critical illness. This is less pronounced in both red cells and white cells. Therefore, intracellular PLP concentrations are more likely to be a reliable measure of status than are plasma measurements in the critically ill patient.

	ACKNOWLEDGMENTS

 
We thank Pamela Moyes, Karen Elliot, Allison McLaughlin, and Lesley Stuart for their assistance and the Scottish Reference Laboratory for Vitamins and Trace Elements, Glasgow Royal Infirmary.

The author's responsibilities were as follows—DCM, JK, AD, DSJO, and DT conceived the idea and funded the study; ATV and JK obtained the consent of the patients and collected the blood samples; ATV and DT prepared and analyzed the blood samples; ATV and DCM performed the statistical analysis; all authors contributed to the drafts and final version of the paper and are the guarantors. None of the authors had a personal or financial conflict of interest.

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