Assessment and clinical implications of functional vitamin B6 deficiency
Minovic, Isidor
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Status and Long-Term
Mortality in Renal
Transplant Recipients
Isidor Minović1,2,3 Anna van der Veen3 Martijn van Faassen3 Ineke J. Riphagen3 Else van den Berg1 Claude van der Ley3
António W. Gomes-Neto1 Johanna M. Geleijnse4 Manfred Eggersdorfer5 Gerjan J. Navis1 Ido P. Kema3 Stephan J.L. Bakker1,2
1Department of Internal Medicine, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands; 2Top Institute Food and Nutrition, Wageningen, the Netherlands; 3Department of Laboratory Medicine, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands; 4Division of Human Nutrition, Wageningen University, Wageningen, the Netherlands; 5DSM Nutritional Products, Kaiseraugst, Switzerland.
Abstract
Background: Low plasma concentrations of pyridoxal 5′-phosphate (PLP)
are common in renal transplant recipients (RTRs) and confer increased risk of long-term mortality. To our knowledge, it is not known whether low plasma PLP concentrations have functional (i.e., intracellular) consequences and, if so, whether such consequences are associated with increased risk of mortality.
Objectives: We assessed the association of plasma PLP with functional
vitamin B6 status and explored the potential association of functional vitamin B6 status with long-term mortality in RTRs.
Design: In a longitudinal cohort of 678 stable RTRs with a median
follow-up of 5.3 y (IQR: 4.8–6.1 y) and 297 healthy controls, PLP, plasma 3-hydroxykynurenine (3-HK), and xanthurenic acid (XA) were analyzed via validated assays. PLP was used as direct biomarker for vitamin B6 status, and the 3-HK:XA ratio was used as functional biomarker of vitamin B6 status with a higher ratio reflecting worse functional vitamin B6 status.
Results: Median PLP, 3-HK, and XA concentrations were 41 nmol/L (IQR:
29–60 nmol/L), 40.1 nmol/L (IQR: 33.0–48.0 nmol/L), and 19.1 nmol/L (IQR: 14.5–24.9 nmol/L), respectively, in healthy controls compared with 29 nmol/L (IQR: 17–50 nmol/L), 61.5 nmol/L (IQR: 45.6–86.5 nmol/L), and 25.5 nmol/L (IQR: 17.2–40.0 nmol/L), respectively, in RTRs (all P < 0.001). RTRs had a higher median 3-HK:XA ratio (2.38; IQR: 1.68–3.49) than did healthy controls (2.13; IQR: 1.63–2.71) (P < 0.05). In RTRs, the 3-HK:XA ratio was inversely associated with plasma PLP (β = −0.21, P < 0.001). Moreover, a higher 3-HK:XA ratio was independently associated with increased risk of all-cause mortality (HR per SD increment: 1.30; 95% CI: 1.13, 1.49), cancer mortality (HR per SD increment: 1.47; 95% CI: 1.12, 1.95), and infectious disease mortality (HR per SD increment: 1.50; 95% CI: 1.21, 1.86) in RTRs.
Conclusions: Vitamin B6–deficient RTRs have a worse functional vitamin
B6 status than do healthy controls and vitamin B6–sufficient RTRs. Worse functional vitamin B6 status in RTRs is independently associated with an increased risk of mortality particularly because of cancer and infectious disease.
Introduction
Vitamin B6 deficiency is common in renal transplant recipients (RTRs) (1–3). Moreover, this deficient state is clinically relevant because it has been shown to confer increased risk of long-term mortality (3). Further substantiating the clinical relevance of vitamin B6 deficiency, studies have established a link between low vitamin B6 status and cancer (4, 5). In addition, vitamin B6 has been suggested to affect the susceptibility to infection in both animal and human studies (6).
Vitamin B6 status is routinely assessed via the plasma concentration of the biochemically important B-6 isoform pyridoxal 5′-phosphate (PLP) (7). However, plasma PLP is affected by various factors that are associated with disease states, including inflammation (8). Consequently, low plasma PLP concentrations may not necessarily indicate true vitamin B6 deficiency. To identify true vitamin B6 deficiency, an additional assessment of functional biomarkers of vitamin B6 is necessary (9), especially in populations marked by inflammation such as RTRs. Functional biomarkers of vitamin B6 status that have been suggested to indicate intracellular PLP availability include kynurenine catabolites of tryptophan, which are converted by enzymes whose biochemical activities depend on the availability of the cofactor PLP (9). In particular, the ratio between 3-hydroxykynurenine (3-HK) and xanthurenic acid (XA) has been suggested as a promising indicator of functional vitamin B6 deficiency.
In the current study, we measured PLP and assessed the 3-HK:XA ratio in healthy volunteers and RTRs and investigated their association in the two groups. In addition, we assessed whether the functional marker of vitamin B6 status (i.e., the 3-HK:XA ratio as putative reflection of intracellular PLP availability) is associated with long-term mortality in RTRs with specific regard to mortality that is due to cancer and infectious disease.
Methods
Study population
This prospective cohort study was based on a previously described, well-characterized set of 707 RTRs and 297 living kidney donors (3, 10). For the participant flowchart, see Supplemental Figure 1. Briefly, this cohort included RTRs (aged ≥18 y) who visited the outpatient clinic of the University Medical Center Groningen, Groningen, Netherlands, between November 2008 and
June 2011 and had a graft that had been functioning ≥1 y with no history of alcohol or drug addiction. We excluded subjects with missing biomaterial (20 cases) and subjects who were taking vitamin B6 supplementation (9 cases) from the statistical analyses, which resulted in 678 cases who were eligible for analyses. We also included 297 healthy kidney donors as a control group, of whom none had to be excluded because of missing biomaterial or vitamin B6 supplementation. The study protocol was approved by the University Medical Center Groningen Institutional Review Board (METc 2008/186) and adhered to the Declaration of Helsinki and Declaration of Istanbul. This trial was registered at clinicaltrials.gov as NCT02811835.
Data collection and measurements
Information on dietary intake was obtained from a validated semiquantitative food-frequency questionnaire (FFQ), which was developed at Wageningen University to assess nutrient intake (11, 12). Because not all participants completed or returned the FFQ, 191 healthy controls and 627 RTRs had data available on dietary intake that were derived from the FFQ, whereas all 297 healthy controls and 678 RTRs had plasma PLP concentrations available. The FFQ inquired about intakes of 177 food items during the past month with seasonal variations taken into account. For each item, the frequency was recorded in times per day, week, or month. The number of servings was expressed in natural units (e.g., slice of bread or apple) or household measures (e.g., cup or spoon). The questionnaire was self-administered and filled out at home. All FFQs were checked for completeness by a trained researcher, and inconsistent answers were verified with the patients. Validation of the FFQ in RTRs was assessed as previously reported (10). Dietary data were converted into daily nutrient intakes with the use of the Dutch Food Composition Table of 2006 (13). The FFQ did not include information on vitamin supplementation. The use of vitamin supplementation by healthy controls and RTRs was recorded separately together with the recording of medication according to patients’ medical records. The use of drugs that might have affected plasma PLP concentrations, including hydralazine (14), oral contraceptives (15), penicillin, dopamine, benzodiazepines, antituberculosis drugs, antiepileptic drugs, and theophyllin (16), were recorded in both healthy controls and RTRs.
Participants were asked to collect a 24-h urine sample on the day before visiting the outpatient clinic. On completion of the 24-h urine collection, fasting blood samples were obtained the next morning, and venous
blood samples were analyzed immediately thereafter. Plasma vitamin B6 was measured as PLP by means of a validated HPLC method (Waters Alliance) with fluorescence detection (Jasco FP-2020; Jasco) (17). Plasma concentrations of 3-HK and XA were obtained with the use of validated liquid-chromatography–tandem mass spectrometry assays (18, 19). The interassay CVs for 3-HK and XA were 9.7% and 2.7%, respectively. Other serum and urine variables were assessed via standard laboratory methods. The glomerular filtration rate was estimated by applying the Chronic Kidney Disease Epidemiology Collaboration equation (20). Diabetes mellitus was diagnosed according to American Diabetes Association criteria as having a fasting plasma glucose concentration ≥7.0 mmol/L or the use of an antidiabetic medication (21). Vitamin B6 deficiency, insufficiency, and sufficiency were defined as plasma PLP concentrations <20, 20–30, and >30 nmol/L, respectively (22).
Clinical endpoints
The primary endpoint of this study was all-cause mortality. Secondary endpoints were cancer mortality, infectious disease mortality, and cardiovascular mortality. Information on the cause of death was derived from the patients’ medical records and was assessed by a nephrologist. Cancer mortality was defined according to a previously specified list of International Classification of Diseases, Ninth Revision (ICD-9) codes (23), whereas infectious disease mortality was identified according to ICD-9 codes 1–13ICD-9. Cardiovascular mortality was defined as death that was due to cerebrovascular disease, ischemic heart disease, heart failure, or sudden cardiac death according to ICD-9 codes 410–447. The continuous surveillance system of the outpatient program ensured that there was up-to-date information on patient status. Endpoints were recorded until September 2015 by a qualified physician. There was no loss that was due to follow-up for the primary endpoints.
Statistical analyses
Data analyses and computations were performed with SPSS 22.0 software (SPSS Inc.), R version 3.2.3 software (The R-Foundation for Statistical Computing), and GraphPad Prism version 5.01 software (GraphPad Software) for Windows (Microsoft).
Baseline data are presented as means ± SDs for normally distributed data, as medians (IQRs) for nonnormally distributed data, and as numbers
(percentages) for nominal data. A 2-sided P < 0.05 was considered to indicate statistical significance.
Differences between healthy controls and RTRs as well as cross-sectional associations of the 3-HK:XA ratio with baseline variables (P-trend) were investigated via univariable linear regression analyses. Multivariable linear regression models were constructed to account for potential confounders such as age, sex, the estimated glomerular filtration rate (eGFR), high-sensitivity C-reactive protein (hs-CRP), and plasma PLP. The percentage of change in the standardized β was calculated as:
[(Standardized β after adjustment
− standardized β before adjustment)
÷ standardized β before adjustment] × 100 (1) In the R software, generalized additive models of the mgcv package (The R-Foundation for Statistical Computing) were used to model the associations of plasma PLP with the 3-HK:XA ratio. The model effect and nonlinearity were tested with the use of 2-sided Wald tests. P-nonlinearity values were calculated by comparing restricted cubic spline terms to linear models. Several subjects had missing values for ≥1 baseline variable [i.e., eGFR, proteinuria, alkaline phosphatase, plasma PLP (<0.5%), smoking status (5.9%), and alcohol intake (8.7%)]. Because the exclusion of subjects with missing values could have resulted in biased prospective results, multiple imputation (fully conditional specification according to the Markov Chain Monte Carlo method) was used to obtain 5 imputed data sets (24, 25). Rubin’s rules were followed to obtain pooled estimates of the regression coefficients and their SEs across the imputed data sets (26).
The prospective associations of the 3-HK:XA ratio with mortality were assessed via Cox regression analyses in which adjustments were made for potential confounders including age, sex, smoking, BMI, eGFR, proteinuria, alkaline phosphatase, hs-CRP, alcohol intake, and plasma PLP. In the longitudinal analyses, the 3-HK:XA ratio was standardized to z values and analyzed as a continuous variable. As a consequence, the HRs of the Cox-regression analyses indicated that there was a relative change in mortality risk per 1-SD change in the 3-HK:XA ratio. Cox regression models were built in a stepwise fashion to avoid overfitting and to keep the number of predictors in proportion to the number of events (27). Potential interactions for covariates were assessed by calculating interaction terms, and the proportionality of hazards was investigated by inspecting the Schoenfeld residuals. To visualize
the continuous associations of the 3-HK:XA ratio with the different types of mortality, the 3-HK:XA ratio, as a continuous variable, was plotted against different risks of mortality. Similar to the generalized additive models, P-nonlinearity values were calculated by comparing restricted cubic-spline terms to corresponding linear models.
Results
Baseline characteristics of the study population
Selected baseline characteristics of the healthy controls and RTRs are presented in Table 1. The median plasma PLP was significantly lower in RTRs (29 nmol/L; IQR: 17–50 nmol/L) than in healthy controls (41 nmol/L; IQR: 29–60 nmol/L) (P < 0.001). Median 3-HK and XA concentrations in RTRs were 61.5 nmol/L (IQR: 45.6–86.5 nmol/L) and 25.5 nmol/L (IQR: 17.2– 40.0 nmol/L), respectively, and 40.1 nmol/L (IQR: 33.0–48.0 nmol/L) and 19.1 nmol/L (IQR: 14.5–24.9 nmol/L), respectively, in healthy controls (P < 0.001). Accordingly, the median 3-HK:XA ratio was higher in RTRs (2.38; IQR: 1.68–3.49) than in healthy controls (2.13; IQR: 1.63–2.71) (β = 0.08, P = 0.02). Adjustment for age, sex, and eGFR increased the standardized β of the comparison of the 3-HK:XA ratio between RTRs and healthy controls to β = 0.31 (both P < 0.001). The difference in 3-HK:XA ratio between healthy controls and RTRs was not markedly affected by additional adjustment for hs-CRP (standardized β = 0.32) and for plasma PLP (standardized β = 0.28). In RTRs, the 3-HK:XA ratio was positively associated with age, hs-CRP, and eGFR (P < 0.01) (Table 2). In contrast, inverse associations were shown between the 3-HK:XA ratio and male sex, alcohol intake, systolic blood pressure, diastolic blood pressure, inorganic phosphate, 24-h urinary creatinine excretion, vitamin B6 intake, total cholesterol, LDL cholesterol, use of antihypertensives, and use of tacrolimus (P < 0.05). After adjustment for age, sex, and eGFR, the association between the 3-HK:XA ratio and systolic blood pressure, inorganic phosphate, 24-h urinary creatinine excretion, vitamin B6 intake, use of antihypertensives, and use of tacrolimus disappeared (P > 0.05). In addition, the same adjustment lowered the standardized β of the association with diastolic blood pressure by 39% to 0.11 (P = 0.002), whereas it revealed an association with HDL cholesterol (standardized β = 0.11, P = 0.003). Additional adjustment for hs-CRP revealed an inverse association between the 3-HK:XA ratio and BMI (standardized β = −0.08, P = 0.03) but left all other associations materially unchanged.
Ta bl e 1. Ba se lin e c ha ra ct er is tic s a nd b io m ark ers o f v ita m in B6 in h ea lth y c on tr ol s a nd r en al t ra ns pl an t r eci pi en ts 1,2 H ea lth y C ont ro ls (N=297) RTRs (N=678) C rude Mo de l 1 Mo de l 2 Mo de l 3 St an d. β St an d. β St an d. β St an d. β A ge , ye ar s 54±11 53±13 -0.02 -M ale g en der , n (%) 138 (47) 398 (57) 0.11** -eGFR , mL/min/1.73m 2 91±14 52±20 -0.39*** -H s-CRP , m g/L 1.1 [0.6-2.3] 1.6 [0.7-4.6] 0.11*** 0.04 -Pl asm a P LP , nm ol/L 41 [29-60] 29 [17-50] -0.22*** -0.21*** -0.20*** -3-HK, nm ol/L 40.1 [33.0-48.0] 61.5 [45.6-86.5] 0.38*** 0.03 0.04 0.02 X A, nm ol/L 19.1 [14.5-24.9] 25.5 [17.2-40.0] 0.23*** -0.24*** -0.23*** -0.25*** 3-HK/X A ra tio 2.13 [1.63-2.71] 2.38 [1.68-3.49] 0.08* 0.31*** 0.32*** 0.28*** 1Abs ol ut e va lues a re p res en te d a s m ea n ± s ta nd ar d de vi at io n, m edi an [in ter qu ar tile ra ng e], o r n um ber (p er cen ta ge). Diff er en ces b et w een g ro ups w er e t es te d w ith uni va ria ble a nd m ul tiva ria ble lin ea r r eg res sio n a na lys es, o f w hic h s ta nd ar dize d (s ta nd .) β’ s a re p res en te d (*P<0.05; **P<0.01; ***P<0.001). 2M ode l 1, ad ju ste d f or a ge , sex, a nd eGFR; m ode l 3, a s m ode l 2, addi tio na lly ad ju ste d f or h s-CRP ; m ode l 4, a s m ode l 3, addi tio na lly ad ju ste d f or p la sm a P LP . R TRs, r en al t ra ns pl an t r eci pien ts; eGFR , es tim at ed g lo m er ul ar fi ltra tio n ra te; h s-CRP , hig h-s en sit iv ity C-r eac tiv e p ro tein; P LP , p yr ido xa l 5’-p hos ph at e; 3-HK, 3-h ydr oxy ky nur enin e; X A, xa nt hur enic acid .
<20 20-30 >30 -1.0 -0.5 0.0 0.5 1.0 1.5 P=0.005 P=0.99 P=0.06 Plasma PLP (nmol/L) Log 3 -HK :X A ra tio <20 20-30 >30 -1.0 -0.5 0.0 0.5 1.0 1.5 P=0.02P<0.001P=0.11 Plasma PLP (nmol/L) Log 3 -HK :X A ra tio 0 20 40 60 80 0 1 2 3 4 5 Plasma PLP (nmol/L) 3−HK:XA ratio 0 20 40 60 80 0 1 2 3 4 5 Plasma PLP (nmol/L) 3−HK:XA ratio
Healthy Controls Renal Transplant Recipients
Figure 1. Continuous associations of 3-HK/XA ratio with plasma PLP and 3-HK/ XA ratios according to clinical categories of plasma PLP in healthy controls (N=297) and renal transplant recipients (N=678)
The variable 3-HK/XA ratio was log transformed for prospective analyses. The histograms illustrate the distribution of plasma PLP. The black lines show the unadjusted hazard ratios
for different types of mortality and the gray areas represent the corresponding 95% CIs. Peffect
were <0.001, 0.02, <0.001, and 0.27 for all-cause mortality (N=144), cancer mortality (N=25), infectious disease mortality (N=41), and cardiovascular mortality (N=57). Corresponding
Pnonlinearity were 0.28, 0.46, 0.28, and 0.22. 3-HK, 3-hydroxykynurenine; XA, xanthurenic acid,
Table 2. Associations of 3-HK/XA ratio with baseline characteristics in 678 renal transplant recipients,1
Crude Model 2 Model 3 Model 4
Demographics Age, years 53±13 0.12** - - -Male gender, n (%) 398 (57) -0.19*** - - -BMI, kg/m2 26.0 [23.2-29.3] -0.02 -0.03 -0.08* -0.09* Smokers, n (%)3 - Never - Past - Current 306 (44) 299 (43) 84 (12) 0.090.12 0.060.08 0.090.07 0.090.10 Alcohol intake, g/d 2.6 [0.0-11.2] -0.16*** -0.12** -0.12** -0.08*
Energy intake, kcal/d 2175±637 -0.05 0.005 0.03 0.03
Vegetarian, n (%) 14 (2) 0.01 -0.01 0.01 0.01
SBP, mmHg 136±18 -0.10* -0.06 -0.05 -0.05
DBP, mmHg 83±11 -0.18*** -0.11** -0.11** -0.12**
Pulse pressure, mmHg 53±13 0.02 0.01 0.03 0.03
Dialysis vintage, months 24 [10-47] 0.05 0.03 0.03 0.01
Time since Rtx, years 5.4 [1.9-12.0] 0.04 -0.001 -0.01 0.03
Plasma PLP, nmol/L 29 [17-50] -0.21*** -0.23*** -0.20***
-Alkaline phosphatase, U/L 67 [54-83] 0.02 0.04 0.01 -0.07
Inorganic phosphate, mmol/L 0.96±0.21 -0.09* -0.04 -0.04 0.02
Creatinine excretion, mg/24h 1287 [1038-1589] -0.16*** -0.05 -0.05 -0.04 Dietary intake Total protein, g/d - Animal protein, g/d - Vegetable protein, g/d 82±20 52±15 31±10 -0.06 -0.05 -0.04 -0.04 -0.05 0.001 -0.03 -0.05 0.03 -0.02 -0.05 0.04 Tryptophan, mg/d 1057±290 -0.08 -0.05 -0.04 -0.03 Fruit, g/d 123 [62-232] 0.04 0.006 0.02 0.05 Vegetables, g/d 91 [52-122] 0.008 -0.002 0.01 0.02 Potassium excretion, mmol/24h 72.6±23.9 -0.07 -0.08* -0.06 -0.03 Vitamin B6 intake, mg/d 1.77±0.48 -0.08* -0.05 -0.03 -0.01 Glucose homeostasis Diabetes, n (%) 170 (24) -0.001 -0.03 -0.06 -0.07* Glucose, mmol/L 5.3 [4.8-6.0] -0.06 -0.08* -0.10** -0.12** HbA1c, % 6.0±0.8 0.02 -0.02 -0.05 -0.07 Tryptophan catabolites 3-HK, nmol/L 61.7 [45.6-86.7] 0.19*** 0.41*** 0.39*** 0.35*** XA, nmol/L 25.6 [17.1-40.1] -0.68*** -0.77*** -0.79*** -0.81*** Inflammation Hs-CRP, mg/L 1.6 [0.7-4.6] 0.16*** 0.17*** - -Leucocytes, 10E9/L 7.7 [7.5-7.9] -0.06 -0.06 -0.08* -0.09*
Table 2. Associations of 3-HK/XA ratio with baseline characteristics in 678 renal transplant recipients,1 (Continued)
Crude Model 2 Model 3 Model 4
Lipids
Total cholesterol, mmol/L 5.1±1.1 -0.10** -0.12** -0.13** -0.11**
HDL-cholesterol, mmol/L 1.4±0.5 0.005 -0.11** -0.08* -0.04 LDL-cholesterol, mmol/L 2.9 [2.3-3.5] -0.11** -0.10** -0.12** -0.11** Triglycerides, mmol/L 1.7 [1.3-2.3] -0.02 0.03 0.01 -0.02 Kidney function eGFR, mL/min/1,73m2 52±20 0.23** - - -Proteinuria, n (%) 158 (23) -0.03 0.02 0.03 0.02 Non-immunosuppressive drugs, n (%) Hydralazine 1 (0.1) 0.04 0.06 0.06 0.06 Benzodiazepines 21 (3) 0.03 -0.001 0.003 0.02 Oral contraceptives 3 (0.4) -0.07 -0.06 -0.06 -0.06 Antiepileptic drugs 8 (1) 0.06 0.04 0.03 0.04 Antihypertensives 613 (88) -0.09* -0.05 -0.06 -0.06 Antidiabetics 110 (16) 0.001 -0.02 -0.04 -0.06 Statins 367 (53) 0.02 -0.002 0.02 0.02 Immunosuppressive drugs, n (%) Calcineurin inhibitor -Cyclosporine -Tacrolimus 274 (39)124 (18) -0.06-0.10* -0.04-0.03 -0.03-0.03 -0.05-0.03 Proliferation inhibitor -Azathioprine -Mycophenolate mofetil 121 (18)454 (66) -0.050.05 -0.070.06 -0.08*0.07 -0.050.03 mTOR inhibitor -Everolimus -Sirolimus 11 (2)13 (6) 0.010.05 0.020.03 0.010.04 0.020.04 Prednisolone 688 (99) -0.03 0.005 0.01 0.004
Absolute values are presented as mean ± standard deviation, median [interquartile range], or number (percentage). Associations between 3-HK/XA ratio and baseline variables were tested with univariable and multivariable linear regression analyses, of which standardized β’s are
presented (*P<0.05; **P<0.01; ***P<0.001). 1Model 1, univariable regression model; model 2,
as model 1, adjusted for age, sex, and eGFR; model 3, as model 2, additionally adjusted for hs-CRP; model 4, as model 3, additionally adjusted for plasma PLP. SBP, systolic blood pressure; DBP, diastolic blood pressure; PLP, pyridoxal 5’-phosphate; 3-HK, 3-hydroxykynurenine; XA, xanthurenic acid; hs-CRP, high-sensitivity C-reactive protein; eGFR, estimated glomerular filtration rate; mTOR, mechanistic target of rapamycin.
Furthermore, additional adjustment for plasma PLP increased the standardized β of the association of the 3-HK:XA ratio with alcohol intake by 33% to −0.08 (P = 0.03), whereas it lowered the standardized βs of the associations with HDL cholesterol and use of azathioprine by 48% and 31%, respectively, to 0.04 (P = 0.37) and −0.05 (P = 0.21), respectively.
The associations between plasma PLP and the 3-HK:XA ratio in RTRs and healthy controls are presented in Figure 1. In both RTRs and healthy controls, plasma PLP concentrations were inversely associated with the 3-HK:XA ratio (P < 0.001 and P = 0.005, respectively). When analyzed according to categories of plasma PLP, vitamin B6–deficient RTRs (i.e., those with plasma PLP concentrations <20 nmol/L) had significantly higher median 3-HK:XA ratios (2.75; IQR: 1.94–4.53) than did vitamin B6 sufficient individuals (2.25; IQR: 1.55–2.96) [i.e., subjects with plasma PLP concentrations >30 nmol/L (P < 0.001)] (Figure 1). A similar difference in 3-HK:XA ratios was shown between vitamin B6–deficient (2.53; IQR: 1.99–3.47) and –sufficient (2.01; IQR: 1.53–2.60) healthy controls (P = 0.005).
Functional vitamin B6 status and mortality
In prospective analyses with a median follow-up of 5.3 y (IQR: 4.8–6.1 y), 144 (21%) of 678 RTRs died, of whom 25 individuals (17%) died because of cancer, 41 individuals (28%) died because of an infectious disease, and 57 individuals (40%) died because of cardiovascular cause. The associations of the 3-HK:XA ratio as a continuous variable with all-cause mortality, cancer mortality, infectious disease mortality, and cardiovascular mortality are shown in Figure 2. The 3-HK:XA ratio was associated with all-cause mortality, cancer mortality, and infectious disease mortality, but not with cardiovascular mortality, in a linear fashion (P-nonlinearity = 0.28, 0.28, and 0.46, respectively). Accordingly, univariable Cox regression analyses revealed significant HRs for the associations between the 3-HK:XA ratio and all-cause mortality (HR: 1.30; 95% CI: 1.13, 1.49), cancer mortality (HR: 1.47; 95% CI: 1.12, 1.95), and infectious disease mortality (HR: 1.50; 95% CI: 1.21, 1.86) (Table 3). These associations remained consistently present, independent of adjustment for potential confounders including age, sex, smoking, BMI, eGFR, proteinuria (model 2), alkaline phosphatase, hs-CRP (model 3), alcohol intake (model 4), and plasma PLP (model 5). Furthermore, the 3-HK:XA ratio was not independently associated with cardiovascular mortality (HR: 1.12; 95% CI: 0.88, 1.43). We showed no significant interaction terms for any covariate.
1 2 3 4 5 6 0.0 0.5 1.0 1.5 2.0 2.5 All−Cause Mor tality 3−HK:XA r atio Risk of mortality 0 10 20 30 Frequency 1 2 3 4 5 6 0.0 0.5 1.0 1.5 2.0 2.5 Cancer Mor tality 3−HK:XA r atio Risk of mortality 0 10 20 30 Frequency 1 2 3 4 5 6 0.0 0.5 1.0 1.5 2.0 2.5 Inf
ectious Disease Mor
tality 3−HK:XA r atio Risk of mortality 0 10 20 30 Frequency 1 2 3 4 5 6 0.0 0.5 1.0 1.5 2.0 2.5 Car dio vascular Mor tality 3−HK:XA r atio Risk of mortality 0 10 20 30 Frequency Fi gur e 2. Th e c on tin uo us a ss oci at io ns o f 3-HK/X A r at io w ith diff er en t t yp es o f m or ta lit y in r en al t ra ns pl an t r eci pi en ts Th e va ria ble 3-HK/X A ra tio wa s log t ra nsf or m ed f or p ros pe ct iv e a na lys es. Th e hi stog ra m s i llu stra te t he di str ib ut io n o f p la sm a P LP . Th e b lac k lin es s ho w t he un ad ju ste d haza rd ra tios f or diff er en t t yp es o f m or ta lit y a nd t he g ra y a re as r ep res en t t he co rr es po ndin g 95% CI s. P effe ct w er e <0.001, 0.02, <0.001, a nd 0.27 f or a ll-c au se m or ta lit y (N=144), c an cer m or ta lit y (N=25), inf ec tio us di se as e m or ta lit y (N=41), a nd c ar dio va sc ul ar m or ta lit y (N=57). C or res po ndin g P non lin ea rit y w er e 0.28, 0.46, 0.28, a nd 0.22. 3-HK, 3-h ydr oxy ky nur enin e; X A, xa nt hur enic acid , P LP , p yr ido xa l 5’-p hos ph at e.
Ta bl e 3. C ox r eg res si on a na ly ses f or t he a ss oci at io n o f 3-HK/X A r at io w ith m or ta lit y in r en al t ra ns pl an t r eci pi en ts (N=687) A ll-c au se m or ta lit y C an cer m or ta lit y In fe ct io us di se as e m or ta lit y C ar di ov as cu lar m or ta lity Nev ent s /Nto ta l 144/678 25/678 41/678 57/678 Mo de l HR [95% CI] P Va lu e HR [95% CI] P Va lu e HR [95% CI] P Va lu e HR [95% CI] P Va lu e 1 1.30 [1.13,1.49] <0.001 1.47 [1.12,1.95] 0.006 1.50 [1.21,1.86] <0.001 1.12 [0.88,1.43] 0.36 2 1.34 [1.16,1.55] <0.001 1.41 [1.04,1.92] 0.03 1.57 [1.24,1.98] <0.001 1.19 [0.92,1.54] 0.20 3 1.30 [1.12,1.51] 0.001 1.38 [1.01,1.89] 0.04 1.57 [1.23,2.01] <0.001 1.11 [0.85,1.46] 0.44 4 1.30 [1.12,1.51] 0.001 1.38 [1.01,1.88] 0.04 1.59 [1.24,2.04] <0.001 1.11 [0.83,1.47] 0.49 5 1.27 [1.09,1.49] 0.002 1.42 [1.04,1.96] 0.03 1.56 [1.20-2.02] 0.001 1.06 [0.78,1.41] 0.75 M ode l 1, cr ude m ode l; m ode l 2, ad ju ste d f or a ge , s ex, sm ok in g, BMI, eGFR , a nd p ro tein ur ia; m ode l 3, a s m ode l 2, addi tio na lly ad ju ste d f or a lka lin e p hos ph at as e a nd hs-CRP ; m ode l 4, a s m ode l 3, addi tio na lly ad ju ste d f or a lco ho l in ta ke; m ode l 5, a s m ode l 4, addi tio na lly ad ju ste d f or p la sm a P LP . 3-HK, 3-h ydr oxy ky nur enin e; X A, xa nt hur enic acid .
Discussion
In a large cohort of stable, outpatient RTRs, we showed that RTRs had a significantly worse functional vitamin B6 status than that of healthy controls. In RTRs, a worse functional vitamin B6 status was associated with low plasma PLP independent of inflammation. A worse functional vitamin B6 status was independently associated with an increased risk of mortality in RTRs, in particular mortality due to cancer and infectious disease. To the best of our knowledge, this is the first prospective observational study that assessed functional vitamin B6 status in RTRs and linked this with long-term mortality.
Analogous to plasma PLP (3), the 3-HK:XA ratio was associated with inflammation, which invoked the hypothesis that the relation between circulating PLP and intracellular PLP concentrations could be influenced by inflammation in RTRs. However, the fact that the association between plasma PLP and the 3-HK:XA ratio was independent of inflammation implies that low circulating PLP indeed reflects low functional vitamin B6 status. Remarkably, we showed a weak association between vitamin B6 intake and functional vitamin B6 status, which disappeared after adjustment for potential confounders. The association between vitamin B6 intake and functional vitamin B6 status may be weak because of a combination of several factors including uncertainty that is associated with the FFQ-derived vitamin B6 estimate, a potential inaccuracy in the food-composition database, and the highly variable bioavailability of vitamin B6 in foods, particularly plant foods in which the bioavailability of vitamin B6 is adversely affected by the presence of fibers and, most importantly, the glucoside conjugate of vitamin B6 pyridoxine glucoside (28). The weak association between vitamin B6 intake and functional vitamin B6 status raises the question as to whether a low functional vitamin B6 status can be corrected only through dietary interventions. However, in patients with coronary artery disease, Ulvik et al. (9) showed that supraphysiologic daily supplementation with 40 mg pyridoxine resulted in ≤50% lower plasma 3-HK:XA ratios and, thus, improved functional vitamin B6 status. These positive effects of vitamin B6 supplementation on functional vitamin B6 status are encouraging, but their potentially beneficial long-term effects remain to be investigated.
Similar to the previously observed prospective associations for plasma PLP (3), we showed that higher 3-HK:XA ratios, as a reflection of worse functional vitamin B6 status, were associated with increased risk of all-cause mortality in RTRs. This association was independent of the plasma PLP
concentration. Although plasma PLP was associated with cardiovascular mortality, we showed no such association for the 3-HK:XA ratio. Alternatively, the 3-HK:XA ratio was independently associated with increased risk of cancer mortality, implying that the 3-HK:XA ratio reflects an essentially different pathophysiology than that of plasma PLP, at least in RTRs. The involvement of vitamin B6 in cancer has recently been suggested by a meta-analysis of 121 observational studies that established associations between low vitamin B6 intake and plasma PLP concentrations and various types of cancer, notably gastrointestinal carcinomas (4). The fact that the observed associations were strongest for gastrointestinal carcinomas might provide important clues as to the mechanisms underlying the observed associations with cancer mortality. Although mechanistic data in this area are scarce, the gut microbiome could be interesting to consider because it has been suggested as an enteric source of vitamin B6 (29). Future studies are needed to unravel the involvement of the gut microbiome in the context of vitamin B6. Analogous to the results of the meta-analysis, the prospective study of Gylling et al. (5) in participants from 2 population-based cohorts showed that low plasma PLP concentrations and high 3-HK:XA ratios, as a reflection of worse functional vitamin B6 status, were associated with increased risk of colorectal cancer. Although a potential relation between vitamin B6 and cancer may be affected by inflammation, this possibility has not been evaluated to our knowledge. In the present study, adjustment for hs-CRP did not materially affect the association between the 3-HK:XA ratio and cancer mortality, thereby suggesting that vitamin B6 might be primarily involved in the carcinogenesis process.
In addition to cancer mortality, we showed that low functional vitamin B6 status was independently associated with infectious disease mortality. A role for vitamin B6 in the defense against infections has been suggested by experimental studies, which showed reduced lymphocyte proliferation (30) and an altered cytokine profile (31) in vitamin B6 deficient rodents. Specifically, a recent study in mice comprehensively showed that vitamin B6 deficiency is able to influence the immune system in 3 ways (i.e., through downregulation of the expression of the suppressor of cytokine signaling 1 protein and upregulation of the immune-cell activator T-bet, through the suppression of T-lymphocyte proliferation, and through decreased secretion of the proinflammatory cytokine IL-2 and increased secretion of the anti-inflammatory cytokine IL-4) (32). Although it is unknown to what extent these data translate to humans, note that the immune system is critical in preventing both infection and cancer, and thus, it is conceivable
that inadequate functional vitamin B6 status by potentially impairing the immune response could confer increased risk of cancer. As a corollary, the potential cross-links between vitamin B6, the human immune system, and carcinogenesis should deserve attention in future studies.
This study has several limitations. First, the observational study design inherently precludes conclusions on causality. Second, the availability of one functional marker of vitamin B6 status makes it difficult to distinguish whether the observed associations were attributable to low intracellular PLP availability or whether they were, e.g., consequences of the biological effects of the separate tryptophan catabolites. Third, we did not have information on the type of cancer or infectious disease that lead to mortality, which limited the discussion of potential mechanisms underlying the observed prospective associations. Fourth, the absence of data on immunologic variables or tumor markers obstructed an extensive evaluation of the potential interplay between vitamin B6, the immune system, and cancer. Strengths of this study include the relatively large study population of RTRs, the long follow-up, the presence of appropriate controls, and the availability of fasting plasma samples. The standardized and high-quality sample-handling procedures and analytic techniques contributed positively to the reliability of our data. Moreover, the well-characterized nature of our cohort allowed for the correction of many factors that could affect vitamin B6 status such as inflammation, medication, and nutrition.
In conclusion, we show that RTRs have a significantly worse functional vitamin B6 status than do healthy controls and that, in both populations, low plasma PLP concentrations are associated with worse functional vitamin B6 status. In RTRs, a worse functional vitamin B6 status is independently associated with increased risk of mortality, particularly mortality that is due to cancer and infectious disease. Our data justify a further characterization of the clinical consequences of functional vitamin B6 deficiency and the potential benefit of the correction thereof in RTRs.
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