Clinical use of cyclooxygenase inhibitors impair vitamin B6 metabolism [Running head: NSAIDs impair vitamin B6 metabolism]
Hsin-Yueh Chang1, Feng-Yao Tang2, Der-Yuan Chen3, Hui-Min Chih1,4, Shih-Ting Huang1, Hung-Dian Cheng1, Joung-Liang Lan3, and En-Pei Isabel Chiang1,5,6*
1Department of Food Science and Biotechnology, National Chung Hsing University, Taichung,
Taiwan, ROC (HYC, HMC, STH, HDC, EPC*)
2Biomedical Science Laboratory, Department of Nutrition, China Medical University, Taichung,
Taiwan, ROC (FYT)
3Division of Allergy Immunology Rheumatology, Taichung Veterans General Hospital,
Taichung, Taiwan, ROC (DYC, JLL)
4Department of Nursing and Pediatrics, Taichung Veterans General Hospital, Taichung, Taiwan,
ROC (HMC)
5NCHU-UCD Plant and Food Biotechnology Program and Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan, ROC (EPC)
6Agricultural Biotechnology Center (ABC), National Chung Hsing University, Taichung, Taiwan,
ROC (EPC)
*Correspondence and requests for reprints should be addressed to En-Pei Chiang [email protected]
Department of Food Science and Biotechnology, National Chung Hsing University 250 Kuo-Kuang Road, Taichung, Taiwan 402, R.O.C
Tel: 886-4-22840385 ext 2190 Fax: 886-4-22876211 Funding sources
This project was supported in part by National Science Council of Taiwan (NSC100-2628-B005-002MY4, NSC101-2320-B-005-006-MY3, NSC 101-2911-I-005-301 to Chiang EP), the
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ATU plan of the Ministry of Education of Taiwan (Chiang EP), the department of health of Taiwan (DOH97-TD-D-113-97011 to Chiang EP) and TCVGH-NCHU 997608 (Chiang EP). Registration information for the study
NCT00944866. Effects of Cox-II Inhibitor on Biochemical Markers in Cardiovascular-related Adverse Effects http://clinicaltrials.gov/show/NCT00944866
PubMed indexing
Chang HY, ChenDY, Chih HM, TangFY, HuangST, ChengHD, LanJL, and Chiang EP* List of abbreviations
DMARDs, disease-modifying antirheumatic drugs; HAQ, Health Assessment Questionnaire; NSAID, non-steroid anti-inflammatory drugs; COX, cyclooxygenase; PLP, pyridoxal
5’-phosphate; PMP, pyridoxamine 5’-5’-phosphate; PNP, pyridoxine 5’-5’-phosphate; PL, pyridoxal; PN, pyridoxine; PM, pyridoxamine; PA, 4-pyridoxic acid; PDXK, pyridoxine kinase; PM(N)PO, pyridoxamine(pyridoxine)-5’- phosphate oxidase; PDXP, Pyridoxal-5'-phosphate phosphatase; PBMCs, peripheral blood mononuclear cells;
Group designations:
RA-COX, non-COX inhibitor users; RA+COX<6 mo, subjects who used COX inhibitor for less than 6 months; RA+COX>6, subjects who used COX inhibitor for more than 6 months.
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Abstract
Background. A low circulating vitamin B6 level, an independent risk factor for cardiovascular disease, is commonly seen in human inflammation. The Objective of the study was to
investigate whether cyclooxygenase (COX) inhibitors alter vitamin B6 metabolism. Designs. To investigate whether subjects on COX inhibitor had altered vitamin B6 profile, a cross-sectional study involving 150 rheumatoid arthritis patients, with and without COX inhibitor treatments, was conducted. C57BL/6J mice and hyperlipidemic Syrian hamsters received drug regimens that reflected clinical non-steroid anti-inflammatory drug (NSAID) uses in treating human inflammation. The impact of long-term physiological use of selective and nonselective COX inhibitors on vitamin B6 metabolism was systematically investigated in these
independent in vivo models. Results. Patients on COX inhibitors had lower circulating pyridoxal-5’-phosphate, especially those taking NSAIDs for more than 6 months. Long-term celecoxib and naproxen use reduced hepatic pyridoxal-5’-phosphate in mice. Nonselective COX inhibitor naproxen significantly decreased B6 vitamers in the kidney. Conclusions. We
demonstrate novel findings that long-term physiological doses of COX inhibitor use may impede the synthesis of coenzymatically active form of vitamin B6. Since the etiology of vitamin B6 depletion in inflammation remains unknown, this study provides a potential mechanism that could account for the poor vitamin B6 status in human inflammation.
Moreover, this study further raises concerns about long-term clinical use of anti-inflammatory NSAIDs in humans. Vitamin B6 status should be carefully monitored in long-term NSAID users. Future randomized placebo control studies are needed to determine the impacts of anti-inflammatory COX inhibitor use on vitamin B6 metabolism in humans.
Key words.
Inflammation; non-steroid anti-inflammatory drugs; cyclooxygenase-2 inhibitor; vitamin B6
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Introduction
The rate-limiting step in arachidonate metabolism is mediated by enzymes known as cyclooxygenases (COXs). These enzymes catalyze the biosynthesis of the precursor of
prostaglandins, prostacyclin and thromboxanes. Nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) and selective COX-2 inhibitors are widely used for symptom relief in human inflammation. Prolonged NSAID use is not rare in patients suffering from incurable diseases such as osteoarthritis and rheumatoid arthritis [1]. The popularity and potential benefits of NSAIDs have encouraged more research on NSAIDs. Accumulating evidence indicates that some NSAIDs can suppress human tumorigenesis in the gastrointestinal tracts [2]. Long-term NSAIDs use for >5 years was reported to be protective against Alzheimer’s disease [3].
However, some NSAIDs, including certain selective COX-2 inhibitors, have been related to an increased risk of cardiovascular events [4,5]. In 2004, one of the initial selective COX-2 inhibitors, rofecoxib, was withdrawn from the market due to an excess risk of
myocardial infarctions and strokes. Despite rofecoxib having a lower gastrointestinal toxicity than naproxen, it was found to be associated with an unanticipated five-fold increase in myocardial infarctions compared with naproxen [6], cerebrovascular incident [7], congestive heart failure [8], stroke [9], renal syndromes [10,11] and arterial hypertension [12].
The predominant coenzymatically active form of vitamin B6 -pyridoxal 5’-phosphate (PLP) (Figure 1) is an essential cofactor for more than 100 biochemical reactions. Poor vitamin B6 status leads to numerous adverse consequences because of its crucial role in the human body. Vitamin B6 deficiency is often present in humans suffering from rheumatoid arthritis and other inflammatory conditions [13-15]. In patients with rheumatoid arthritis, the concentration of PLP is associated with multiple markers of inflammation, including disease activity and severity, synovial burden, and pain [14]. In an experimental animal model of arthritis, inflammation causes tissue-specific depletion of vitamin B6 [16]. Low plasma vitamin B6 status affects metabolism through the kynurenine pathway in cardiovascular patients with
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systemic inflammation [17]. Abnormal vitamin B6 status in rheumatoid arthritis has been recognized for decades, but the etiology remains to be established [13-15,18]. Reduced levels of vitamin B6 confer an increased risk of atherosclerosis in humans [19], and vitamin B6 deficiency induced arteriosclerotic lesions in an animal model [20]. Low plasma PLP levels are independently associated with increased risk of coronary artery disease [21]. Combined with the chronic inflammatory condition that has been proved atherogenic [22], a lower plasma PLP level [19] in patients with rheumatoid arthritis could further promote cardiovascular diseases in these patients [23]. In fact, patients with rheumatoid arthritis have twice the probability of cardiovascular death compared with the general population [24]. Therefore, a well-chosen anti-inflammatory therapy for these patients will have implications in many regards.
Conventional NSAIDs and selective COX-2 inhibitors are widely prescribed in patients with inflammation, but data on their effects on vitamin B6 status are limited. The present study investigated whether chronic suppression of COX activity through clinical use of NSAIDs or COX-2 inhibitor would interfere with vitamin B6 metabolism. The cross-sectional study provided the association between altered B6 profile and clinical NSAIDs use in humans. The in
vivo experimental models enabled us to study how physiological doses of COX inhibitors may
affect vitamin B6 metabolism in the tissues under well-controlled conditions.
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Designs and Methods
Chemicals
Pyridoxal-5’-phosphate (PLP), 4-pyridoxic acid (4-PA), pyridoxal hydrochloride (PL), pyridoxamine-5’-phosphate (PMP) and all other chemicals were purchased from Sigma (St. Louis, MO, USA). HPLC-grade methanol was purchased from Echo (Miaoli, Taiwan). Celecoxib and naproxen were obtained as commercial drugs: Celebrex® (Pfizer, New York, USA) and GenuproxinTM (Genuine, Taoyuan, Taiwan).
Human study protocol
The study protocol was approved by the institutional clinical research ethics committee. A total of 150 adults (age 18 years and over) with rheumatoid arthritis meeting the American College of Rheumatology criteria for rheumatoid arthritis [25]were recruited prospectively from 2008-2010 at the Allergy, Immunology and Rheumatology outpatient clinic at the Department of Immunology and Rheumatology, Taichung Veterans General Hospital (TCVGH), Taiwan. Written informed consents were obtained from all subjects prior to enrollment in accordance with the regulation of the Institution. Subjects with pregnancy, anemia (hemoglobin 10 mg/dL or lower), thrombocytopenia (platelet count below 50,000 cells/ L), abnormal serum hepatic transaminase (aspartate aminotransferase or alanine μ aminotransferase above 50 IU/L), diabetes or cancer were excluded. Subjects were asked to avoid taking over-the-counter aspirin and vitamin supplements for at least 1 month prior to enrollment in the study. Blood chemistry analyses were performed at the general clinical research lab at TCVGH that included, creatinine, CRP, white count, platelets, and hemoglobin.
Duration of COX inhibitor use
NSAIDs used in study subjects included: celecoxib, meloxicam, nimed, sulindac and diclofenac. Among those taking COX inhibitors, approximately 74% of the subjects had been on NSAIDs for more than 6 months (mean duration 16 mo). The duration of NSAID use was based on the prescription history that was confirmed by a questionnaire administered by the
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study personnel. Duration of NSAID use was then divided into three categories: no use, ≤6 months of use, >6 months of use.
Human study experimental protocol
Subjects were asked to fast overnight for 12 h for the study blood draw. The following morning, fasting blood samples from the subjects were collected into
ethylenediaminetetraacetic acid tubes and chilled immediately on ice for the determination of vitamin B6 profile and homocysteine. Each patient was then instructed to complete the
Stanford Health Assessment Questionnaire to evaluate how well the patient is able to perform routine activities [26] which has 20 categories of questions that evaluate how well the patient is able to manage routine activity. Assessments of disease activity were rated on a scale of 0 to 3, where 0 indicates remission; 1, mild; 2, moderate; and 3, severe. A visual analog scale was used for assessing the "pain score"; and a tender joint count (28- joint count) [14,26]was performed for each subject by the study physicians.
Gene expression of vitamin B6 metabolic enzymes in human PBMCs
Plasma and RNA samples were isolated and stored at -80°C until analysis. The peripheral blood mononuclear cells (PBMCs) were collected from heparinized blood and isolated by Ficoll-Hypaque centrifugation, then RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA). The quality of all RNA samples was checked on agarose gel.
Complementary DNA (cDNA) was copied from 2 g total RNA using Moloney murine leukemiaμ virus reverse transcriptase (Promega, Madison, WI, USA) primed with oligo(dT)[27]. Equal amounts of cDNA were taken from each reverse transcription (RT) reaction mixture for real-time polymerase chain reaction (PCR) amplification, using specific primers for pyridoxal kinase (PDXK), pyridoxal 5’-phosphate phosphatase (PDXP), pyridoxamine 5’-phosphate oxidase (PM(N)PO) and for 18S as internal controls. Real-time PCR analysis was performed using the SYBR Green PCR Reagents Kit (Applied Biosystems, Foster City, CA, USA).
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Dietary intake of study subjects
Subjects were instructed to complete a food diary at enrollment. Each subject was interviewed by a registered dietitian for validation of the food diary on the day of blood collection. A colored book with 1:1 size food photos were used to standardize portion size among subjects. Nutrient composition was calculated with the use of Nutritionist Professional software [27] (E-kitchen Business Corporation, Taipei, Taiwan), in combination with the Taiwan food composition database, established by the Department of Health in Taiwan.
Animals, diet and study conditions
Both animal studies were approved by the Institutional Animal Care and Use Committee. Study 1 investigated the impacts of physiological doses of selective COX II
inhibitor celecoxib and nonselective COX inhibitor naproxen on tissue vitamin B6 status. Study 2 investigated the effects of long-term physiological doses of celecoxib and naproxen
treatments on vitamin B6 metabolism in hypercholesterolemic animals. In Study 1, 18 male 4-week-old C57BL/6J mice (National Laboratory Animal Center, Taipei, Taiwan) were fed a standard AIN93M (Dyets, Bethlehem, PA) that contained 6 mg pyridoxine per kg diet
throughout the study period [28]. In Study 2, 18 male 7-week-old Syrian hamsters (National Laboratory Animal Center, Taipei, Taiwan) were fed a modified AIN-93M hypercholesterolemic diet (10% corn oil, plus 0.2% cholesterol) that contained 6 mg pyridoxine per kg diet
throughout the study period (Dyets, Bethlehem, PA). After 2 weeks of adaptation, animals in both studies were divided by weight into vehicle control, naproxen, and celecoxib groups. All animals were maintained in a temperature- and humidity-controlled environment (20-25OC) with a 12 h light/12 h dark cycle with food and sterilized water ad libitum. The food intakes were measured daily, and body weight was recorded twice a week. A 24-h urine specimen was collected from each animal; excretion of urinary 4-pyridoxic acid and creatinine was
subsequently measured by an enzymatic colorimetric assay followed the manufacturer’s instruction (Randox Laboratories Ltd, County Antrim, UK).
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Clinical relevance of celecoxib and naproxen regimens
Based on the life expectancy of the animal models, the duration of Study 1 would reflect 4–5 years; study 2 would reflect more than 10 years of long-term clinical use of these treatments in humans. According to the “Guideline for the timing of nonclinical safety studies for the conduct of human clinical trials for pharmaceuticals” [29], a study treatment
continuing for longer than 10% of the test subject’s life span would be considered “chronic”. Conversion of the animal doses to human equivalent doses was calculated based on body surface area [30]. On the day of the treatment, animals in the treatment groups received celecoxib or naproxen treatments (freshly dissolved in saline) by gastric-feeding at the
beginning of the dark cycle at 18:00. Control animals received the same volume of 0.9% saline as the treatment by gastric-feeding. In Study 1, mice received either naproxen (5 mg naproxen per kg body weight per day), or celecoxib (12.5 mg/kg body weight per day) or saline vehicle every other day for a period of 7 weeks. In Study 2, hamsters received saline, celecoxib (21.2 mg/kg/day) or naproxen (8.5 mg/kg/day) for a total of 22 weeks. The doses used in Study 1 were equivalent to 61 mg celecoxib per person per day (1 mg celecoxib/kg BW) and 24 mg naproxen per person per day (0.4 mg celecoxib/kg BW). The doses used in Study 2 were equivalent to 172 mg celecoxib and 69 mg naproxen per person daily. Celecoxib is generally administered to arthritis patients at a dosage of 200 mg/day; naproxen is generally used at a dosage of 100 mg–200 mg/day. The mean daily dose of celecoxib in our study subjects was 262 mg (ranged between 100 mg–400 mg). The doses used in our studies were well below the doses used in patients suffering from chronic arthritis.
Blood and tissue sample collection and preparation
After the study periods, fasting blood samples were drawn from the orbital venous sinus vein into heparin-coated tubes. Plasma was separated immediately by centrifugation and stored at -80°C. Animals were sacrificed by exsanguination under deep anesthesia. The liver and kidneys were immediately excised and stored in -80oC until analysis.
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Determination of vitamin B6 and homocysteine concentrations
Plasma was precipitated with 0.4 M ice-cold perchloric acid for deproteinization before HPLC analysis. Animal tissue was homogenized in ten volumes of 0.4 M ice-cold perchloric acid. The extracts were kept on ice for 30 min, then centrifuged at 14000 g for 10 min at 4°C, and the supernatants were frozen in -80°C [31]. The pre-column semicarbazide dramatization followed the procedure previously described [32] with modifications for additional detection of B6 vitamers in tissues. Briefly, PCA extracted tissue supernatants were incubated (30 min in the dark) with derivatization solution (250 mg/ml semicarbazide and glycine). Samples were filtered through 0.45 m microspin filters prior to HPLC analysis μ [28]. The HPLC system consisted of a Hitachi L-2130 intelligent pump connected to an L-2480 fluorescence detector and an L-2100 autosampler (Hitachi, Tokyo Japan). Separation was achieved using a Mightysil RP-18 column (250 × 4.6 mm I.D, particle size 5 m)(Kanto Corp, μ Portland, OR, USA). Mobile phase A consisted of 60 mmol/L disodium hydrogen phosphate containing 9.5% methanol (v/v) and 400 mg/L EDTA disodium salt and adjusted to pH 6.5 with phosphoric acid. Mobile phase B consisted of 60 mmol/L disodium hydrogen phosphate and 400 mg/L EDTA disodium salt, adjusted to pH 6.0 with phosphoric acid. To detect B6 vitamer PMP, the fluorescence detector was programmed the excitation (Ex) and emission (Em) wavelengths at 325 and 400 nm to detect PMP. The intra- and inter-assay CV were both below 10% and the recovery was between 90%-110%. The detailed HPLC procedure will be described elsewhere (Chiang EP, Chang HY, Lin SJ, and Hou HC. unpublished data, 2011). Plasma total (unbound and bound) homocysteine, was measured by HPLC with fluorometric detection was determined by the method described previously [33].
Statistical analysis
Non-normally distributed variables were log-transformed to reach normality for statistical analyses. One-way ANOVA was used for multiple comparisons; and the Bonferroni corrected p values were calculated in post-hoc analyses. Pearson’s correlation coefficients
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were calculated between continuous variables including concentrations of B6 vitamers, gene expression levels of PDXK, PDXP, PM(N)PO and the duration of NSAIDs (in months). Differences were considered significant when p-value was <0.05. All statistical analyses were performed using Systat 11.0 for Windows (Systat Software Inc., Richmond, CA, USA).
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Results
Clinical Characteristics
Health Assessment Questionnaire (HAQ) disability score of the study subjects indicated remission to mild disease activity in the study subjects. The mean (± SD) Health Assessment Questionnaire disability score of the patients (n=147) was 0.43 ± 0.68 (on a 0 to 3 scale), indicating that the conditions were well controlled (Table 1). Mean disease duration was 13.0 (median duration 12.5) years. Of the 150 patients originally enrolled in the study, three subjects dropped out (or were excluded) because of continuing vitamin
supplementation or concerns over discontinuing their vitamin supplements. All patients were taking medications for symptom relief and had been taking the same medications for at least 2 months. Among the 147 patients who completed the study, 26 (18%) were not taking any kind of COX inhibitors; 121 (82%) were taking COX inhibitors (number of users taking each
medication: celecoxib=63; meloxicam=35; sulindac=12, nimed=8; diclofenac=3). Of those 121 COX inhibitor users, 89 (74%) had used NSAIDs for more than 6 months. Subjects were
divided into three groups according to the durations of their COX inhibitor use: non-users (RA-COX, n=26) who did not use any NSAIDs during and at least 2 months prior to the enrollment; those who used for less than 6 months (RA+COX<6 mo, n=32); and those who used for more than 6 months (RA+COX>6 mo, n=89). Clinical and demographic
characteristics of the subjects are shown in Table 1. Distribution of gender, age, body mass index (BMI), disease duration, disease activity and blood chemistry measurements were similar among the 3 groups. The percentage of disease-modifying antirheumatic drug (DMARD) use, treatment duration or weekly dose did not significantly differ among the 3 groups, suggesting that the potential impacts on the biochemical parameters from other drugs were minimal.
Lower vitamin B6 levels were present in patients taking COX inhibitors
Dietary intake of calories, protein, vitamin B6 or vitamin B6 to protein ratio did not
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differ among non-COX-users and COX users (Table 2). In comparison with the general population in the Nutrition and Health Survey in Taiwan (NAHSIT), our subjects had lower mean energy consumption (Current study females: 1212 Kcal, males: 1429 Kcal vs. NAHSIT females: 1459 Kcal, NAHSIT males: 1821 Kcal). These findings were in consistent with a lower prevalence in overweight/obesity (BMI>24) in our subjects (<32%) compared to that (>51%). of the 2005-2008 NAHSIT [34]. Our subjects had similar percent energy from fat (30.2%) compared to NAHSIT (31.4%), more energy from carbohydrate (current study: 56.3 % vs. NAHSIT: 51.7%) but lower energy from protein (current study: 13.5 % vs. NAHSIT: 17%). The mean vitamin B6 intake in our subjects was slightly lower than that of NAHSIT, consistent with the fact that these subjects consumed less protein containing foods that are also common sources for vitamin B6. The lower B6 intake in the study subjects could also be associated with the exclusion of supplement users. The mean B6 to protein intake ratio in our subjects (0.02+/-0.01, n=147) was similar to that of the subjects in the NAHSIT (0.02+/-0.02, n=1911) [34].
Plasma PLP and PL concentrations of the study subjects were in good agreement with rheumatoid arthritis patients in previous studies [14,16,35,36]. Compared to the non-COX users, plasma PLP concentrations tended to be lower in short-term users (RA+COX<6 mo, P<0.1) and were significantly lower in those long-term users (RA+COX>6 mo, p=0.003). Plasma PLP to PL ratio, which may reflect the efficiency to convert B6 into its active form, was significantly reduced in all COX users (Table 2).
On the other hand, Plasma homocysteine concentration or the gene expression of
PDXK, PDXP and PM(N)PO, major metabolic enzymes of vitamin B6 (Figure 1) did not differ
among these groups (Table 2).
Specific COX-2 inhibitor and nonselective NSAID alter vitamin B6 metabolism in vivo
In both mouse and hamster models, mean body weight and food intake did not differ among animals receiving saline, celecoxib or naproxen as the treatment. Plasma creatinine
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levels remained unaffected in these animals throughout the study (data not shown). These results suggested that treatments of celecoxib or naproxen at these physiological doses did not affect animal growth, food consumption, or renal function throughout the study period.
Although plasma PLP or PL concentrations did not change by the 7-week or 22-week point of COX inhibitor treatments in these models (Table 3-4), celecoxib and naproxen
significantly changed the vitamin B6 status in tissues. In mice fed the AIN93M diet, the 7-week celecoxib treatment resulted in a 12% reduction in PLP in the liver; and the 7-week naproxen treatment decreased hepatic PMP and PLP concentrations (Table 3) by 12% and 21%,
respectively. In hypercholesterolemic hamsters, the 22-week-long-term celecoxib and naproxen treatments decreased hepatic PLP concentrations by 30% and 28%, respectively (Table 5). In the kidney, naproxen, but not celecoxib, decreased PLP in both models. In
hamsters receiving 22 wks of NSAID treatments, celecoxib and naproxen drastically decreased PL by 60% and 70%, respectively (Table 5). Since none of these treatments affected dietary intake in the models, the reductions in tissue B6 vitamers observed in these animals were likely caused by the interference of vitamin B6 metabolism and not by reduced vitamin B6 consumption. The 22-week-long-term celecoxib and naproxen treatments tended to decrease the PLP to PL ratio in the liver, and these treatments significantly decreased the PL to PA ratio in the kidney (Table 5).
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Discussion
By demonstrating the novel findings pertaining to the interfering effects of specific NSAID treatments on vitamin B6 metabolism, our present study underscores the need for continued surveillance and pharmacological research of COX inhibitors in clinical practice.
Abnormal vitamin B6 status in rheumatoid arthritis has been recognized for decades, but the basis of the low plasma PLP concentrations in human inflammation remains to be established. In this study, we provide an additional cause that may in part account for the poor vitamin B6 status present in subjects suffering from chronic inflammation. We demonstrated significant findings on the interfering effects of specific long-term NSAID treatments on vitamin B6 metabolism. None of those treatments affect dietary intake in the models,
suggesting that the reductions in B6 vitamers observed in these animals were unlikely due to reduced vitamin B6 consumption, but rather due to the interference in the uptake, retention or utilization of vitamin B6. The effects of celecoxib and naproxen on vitamin B6 status are summarized in Table 5.
The cause of B6 depletion by NSAIDs treatment is unknown. Studies have demonstrated constitutive COX expression in the kidney and the significance of COX-2 in regulating renal function [37-39]. The hypertensive effects of celecoxib may have been
associated with the effects of this drug on the kidney because COX-2 plays a role in regulating renin release, and selective inhibition of COX-2 in the rat results in a reduction in plasma renin activity [40]. Doses of celecoxib in the range used for treatment of rheumatoid arthritis
significantly suppressed urinary excretion of prostacyclin metabolites in healthy volunteers [41]. Many patients with chronic renal failure often develop vitamin B6 deficiency, [42] and have higher vitamin B6 demand [43]. Low plasma PLP levels have been shown in renal disorders [44]. However, uremia is unlikely the main cause of poor vitamin B6 status in these subjects because neither the human subjects nor the animals had abnormal creatinine. Furthermore, the effect of NSAIDs on kidney function appears to be acute and to recede over
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time [45]; yet most of our study subjects had used NSAIDs for more than 1 month. The lower ratio of PLP to PL in human plasma, as well as in the liver of animals receiving long-term celecoxib and naproxen indicated that these drugs may have specifically interfered with the conversion of B6 into its coenzyme form in the major organ of metabolism. Celecoxib and naproxen both resulted in significant reductions of PL in the kidney of hamsters receiving the NSAID treatments for over 20 weeks. In vitamin B6 metabolism, the delivery, transport and uptake of vitamin B6 is mainly in the form of PL in the body [46]. Renal tubules play a critical role in salvaging vitamin B6 from luminal filtrate [47-49]. Experiments showed that the uptake of radioisotope vitamin B6 by freshly isolated rat renal proximal tubular cells was temperature dependent and exhibited saturation kinetics [47]. In vitamin B6 catabolism, PL arising from dephosphorylation of PLP can be irreversibly converted to 4-pyridoxic acid (4-PA) by either an NAD-dependent dehydrogenase or an FAD-dependent aldehyde oxidase [46] (Figure 1). Mean 4-PA excretions appeared to be slightly elevated in animals received long-term COX inhibitors but the alterations did not reach significance. Since the altered PL concentrations were observed in the kidney but not in the liver or the circulation, the significant reductions of PL in the kidney may reflect poor reabsorption of vitamin B6
although more studies are needed for this postulation. How NSAIDs specifically interfere with the inter-conversion of B6 vitamers are currently under investigation.
In patients with rheumatoid arthritis, plasma PLP level is a good diagnostic indicator of functional vitamin B6 status, as it was inversely correlated with the homocysteine increase in response to a methionine load, and also inversely correlated with the xanthurenic acid excretion in response to a tryptophan load [36]. A functional assessment of vitamin B6 status would help determine the biological relevance of the low circulating vitamin B6 level in COX treated humans and animals.The vitamin B6 functional status is under investigation in animal models.
It was reported that mice or hamsters, rather than guinea pigs or rats, would be better
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species for quantitative studies of pyridoxine-glucoside bioavailability and associated enzymatic processes [50]. However, these 2 animal models may not be the best system for studying the drug effects of vitamin B6 metabolism. It remains to be investigated whether these animals are ideal models of what occurs in humans, as the COX inhibitors affected plasma PLP in the humans, but not in the animal models.
Although the results from our human subjects were derived from an observational design, the interference of NSAIDs on vitamin B6 metabolism is reinforced by the consistency of our results in 2 in vivo models. By carefully controlled known factors that might affect the outcomes (dietary B6 contents, age, gender, type and doses of NSAIDs), these models clearly provided a cause-effect relationship between physiological NSAID use and tissue-specific B6 depletion. In hamsters receiving NSAID treatments, celecoxib and naproxen drastically decreased PL by 60%-70%, indicating that long-term use of COX inhibitors, regardless
selective or non-selective COX-2 inhibitors could disrupt vitamin B6 metabolism in the kidney. Because the case number of each individual conventional NSAIDs was low, the
comparison of each drug on vitamin B6 metabolism was not feasible in these human subjects. More studies of NSAIDs on vitamin B6 absorption are needed in this regard. Furthermore, NSAIDs are used primarily by humans suffering from chronic inflammation. This is the same population likely to be deficient in vitamin B6. One would expect that control of their
inflammatory conditions would help improve their vitamin B6 status. However, the results of the present study suggest that use of NSAIDs in the dose range used for treating rheumatoid arthritis may deplete vitamin B6 in important tissues of vitamin B6 metabolism. The effects of conventional NSAIDs and selective COX-2 inhibitors on renal function could result in
detrimental effects on vitamin B6 status. Given the very high coincidence of coronary disease, vitamin B6 deficiency and inflammation, this population may represent the largest portion of the population for whom NSAIDs and selective COX inhibitors are prescribed and the
extensive use of these drugs puts many of them at risk. Selection of anti-inflammatory
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treatment is of particular significance for this population. However, relatively fewer studies have investigated the link between medication use and impaired vitamin B6 status in chronic inflammation. Using two independent in vivo models we recently demonstrated a potential advantage of clinical prednisolone use in treating inflammation with respect to vitamin B6 status [28].
Conclusions
The present study presents new findings that long-term use of COX inhibitor
physiological doses may impede the synthesis of the coenzymatically active form of vitamin B6. Considering that the etiology of vitamin B6 depletion in inflammation remains unknown, this study not only provides an additional cause that could account for the low circulating B6 level in human inflammation but also raises concerns about long-term clinical use of NSAIDs. In light of reports of cardiovascular harm associated with COX inhibitors, our data provide evidence that the use of certain COX inhibitors may increase the risk of cardiovascular diseases via impaired vitamin B6 status. Whether these alterations were due to direct inhibition of the conversion into its coenzyme form, poor reabsorption, or acceleration of excretion remain to be further investigated. Nonetheless, vitamin B6 status should be more carefully monitored in long-term NSAID users. Future randomized placebo control studies are needed to determine the impacts of anti-inflammatory COX inhibitor use on vitamin B6
metabolism in humans. 35 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409
Acknowledgements
The authors thank Hsien-Hua Hsieh, Lan-Ying Lu, Wei-Wen Chen, Chia-Yuen Hsu, and Lai-In Wong for assistance in subject recruiting, dietary assessments, or data entry (Food Science Technology at NCHU). The authors also thank Chih-An Lin, An-Ti Liu, and Po-Hsuan Chiufor assistance in animal experiments (Food Science & Technology at NCHU). Thanks are also given to the staff at the Department of Allergy, Immunology and Rheumatology,
Department of Nursing, and the Clinical Chemistry Laboratory at Taichung Veterans General Hospital for general support.
Competing interests
There is no financial or non- financial competing interest. Authors' contributions
All authors made substantive intellectual contributions to the present study and approved the final manuscript. EPC conceived of the study, generated the original hypothesis, acquired funding, performed statistical analysis and data interpretation, drafted and revised the manuscript. HYC designed the study, obtained clinical samples, performed biochemical measurements, statistical analyses, and literature review. FYT, STH and HDC conducted animal experiments and performed biochemical analyses. DYC and JLL performing clinical
assessments on study subjects. HMC was in charge of subject recruiting, clinical data acquisition and literature review. EPC had primary responsibility for the final content. All authors read and approved the final manuscript.
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