Multicenter Evaluation of Independent High-Throughput and RT-qPCR Technologies for the Development of Analytical Workflows for Circulating miRNA Analysis.

BACKGROUND: Among emerging circulating biomarkers, miRNA has the potential to detect lung cancer and follow the course of the disease. However, miRNA analysis deserves further standardization before implementation into clinical trials or practice. Here, we performed international ring experiments to explore (pre)-analytical factors relevant to the outcome of miRNA blood tests in the context of the EU network CANCER-ID. METHODS: Cell-free (cfmiRNA) and extracellular vesicle-derived miRNA (EVmiRNA) were extracted using the miRNeasy Serum/Plasma Advanced, and the ExoRNeasy Maxi kit, respectively, in a plasma cohort of 27 NSCLC patients and 20 healthy individuals. Extracted miRNA was investigated using small RNA sequencing and hybridization platforms. Validation of the identified miRNA candidates was performed using quantitative PCR. RESULTS: We demonstrate the highest read counts in healthy individuals and NSCLC patients using QIAseq. Moreover, QIAseq showed 15.9% and 162.9% more cfmiRNA and EVmiRNA miRNA counts, respectively, in NSCLC patients compared to healthy control samples. However, a systematic comparison of selected miRNAs revealed little agreement between high-throughput platforms, thus some miRNAs are detected with one technology, but not with the other. Adding to this, 35% (9 of 26) of selected miRNAs in the cfmiRNA and 42% (11 of 26) in the EVmiRNA fraction were differentially expressed by at least one qPCR platform; about half of the miRNAs (54%) were concordant for both platforms. CONCLUSIONS: Changing of (pre)-analytical methods of miRNA analysis has a significant impact on blood test results and is therefore a major confounding factor. In addition, to confirm miRNA biomarker candidates screening studies should be followed by targeted validation using an independent platform or technology.

Multicenter Evaluation of Circulating Cell-Free DNA Extraction and Downstream Analyses for the Development of Standardized (Pre)analytical Work Flows.

BACKGROUND: In cancer patients, circulating cell-free DNA (ccfDNA) can contain tumor-derived DNA (ctDNA), which enables noninvasive diagnosis, real-time monitoring, and treatment susceptibility testing. However, ctDNA fractions are highly variable, which challenges downstream applications. Therefore, established preanalytical work flows in combination with cost-efficient and reproducible reference materials for ccfDNA analyses are crucial for analytical validity and subsequently for clinical decision-making. METHODS: We describe the efforts of the Innovative Medicines Initiative consortium CANCER-ID (http://www.cancer-id.eu) for comparing different technologies for ccfDNA purification, quantification, and characterization in a multicenter setting. To this end, in-house generated mononucleosomal DNA (mnDNA) from lung cancer cell lines carrying known TP53 mutations was spiked in pools of plasma from healthy donors generated from 2 different blood collection tubes (BCTs). ccfDNA extraction was performed at 15 partner sites according to their respective routine practice. Downstream analysis of ccfDNA with respect to recovery, integrity, and mutation analysis was performed centralized at 4 different sites. RESULTS: We demonstrate suitability of mnDNA as a surrogate for ccfDNA as a process quality control from nucleic acid extraction to mutation detection. Although automated extraction protocols and quantitative PCR-based quantification methods yielded the most consistent and precise results, some kits preferentially recovered spiked mnDNA over endogenous ccfDNA. Mutated TP53 fragments derived from mnDNA were consistently detected using both next-generation sequencing-based deep sequencing and droplet digital PCR independently of BCT. CONCLUSIONS: This comprehensive multicenter comparison of ccfDNA preanalytical and analytical work flows is an important contribution to establishing evidence-based guidelines for clinically feasible (pre)analytical work flows.

Multicenter Evaluation of Circulating Plasma MicroRNA Extraction Technologies for the Development of Clinically Feasible Reverse Transcription Quantitative PCR and Next-Generation Sequencing Analytical Work Flows.

BACKGROUND: In human body fluids, microRNA (miRNA) can be found as circulating cell-free miRNA (cfmiRNA), as well as secreted into extracellular vesicles (EVmiRNA). miRNAs are being intensively evaluated as minimally invasive liquid biopsy biomarkers in patients with cancer. The growing interest in developing clinical assays for circulating miRNA necessitates careful consideration of confounding effects of preanalytical and analytical parameters. METHODS: By using reverse transcription quantitative real-time PCR and next-generation sequencing (NGS), we compared extraction efficiencies of 5 different protocols for cfmiRNA and 2 protocols for EVmiRNA isolation in a multicentric manner. The efficiency of the different extraction methods was evaluated by measuring exogenously spiked cel-miR-39 and 6 targeted miRNAs in plasma from 20 healthy individuals. RESULTS: There were significant differences between the tested methods. Although column-based extraction methods were highly effective for the isolation of endogenous miRNA, phenol extraction combined with column-based miRNA purification and ultracentrifugation resulted in lower quality and quantity of isolated miRNA. Among all extraction methods, the ubiquitously expressed miR-16 was represented with high abundance when compared with other targeted miRNAs. In addition, the use of miR-16 as an endogenous control for normalization of quantification cycle values resulted in a decreased variability of column-based cfmiRNA extraction methods. Cluster analysis of normalized NGS counts clearly indicated a method-dependent bias. CONCLUSIONS: The choice of plasma miRNA extraction methods affects the selection of potential miRNA marker candidates and mechanistic interpretation of results, which should be done with caution, particularly across studies using different protocols.

Prevalence of labrum and articular cartilage injuries of the hip on 3T magnetic resonance imaging of asymptomatic elite soccer players. / Prevalencia de lesiones del labrum y del cartílago articular de la cadera en imagen por resonancia magnética simple 3 T de futbolistas profesionales asintomáticos.

OBJECTIVE: To establish the prevalence of lesions of the labrum and articular cartilage of the hip in asymptomatic elite soccer players by performing 3T magnetic resonance imaging. METHODS: Eighty-four asymptomatic hips of 42 professional soccer players were evaluated. Male subjects older than 18 years were included. Cam and pincer deformity were defined as an alpha angle greater than 55 degrees and a lateral centre edge angle greater than 39 degrees, respectively. Labral injuries were classified with the Czerny classification and cartilage damage was classified with the Outerbridge classification. Specific statistical tests were used to establish the relationship between anatomical variances of the hip and the presence of chondral and labral injuries. RESULTS: FAI morphology prevalence was 25%. Abnormalities such as cam (22.5%) and labral injuries (33.8%) were found. Those cases with reported labral injury were predominantly intrasubstance damage (18.8%). Anatomical features of FAI were found to be related to lesions of the femoral cartilage (P<.001), chondrolabral damage (P=.042), or both injuries (P<.001). CONCLUSION: Asymptomatic labral or cartilaginous injuries of the hip were reported in 25% of the included professional soccer players. These injuries were associated with anatomical features of FAI.

ctDNA and CTCs in Liquid Biopsy - Current Status and Where We Need to Progress.

We discuss the current status of liquid biopsy and its advantages and challenges with a focus on pre-analytical sample handling, technologies and workflows. The potential of circulating tumor cells and circulating tumor DNA is pointed out and an overview of corresponding technologies is given.

A Novel Workflow to Enrich and Isolate Patient-Matched EpCAMhigh and EpCAMlow/negative CTCs Enables the Comparative Characterization of the PIK3CA Status in Metastatic Breast Cancer.

Circulating tumor cells (CTCs), potential precursors of most epithelial solid tumors, are mainly enriched by epithelial cell adhesion molecule (EpCAM)-dependent technologies. Hence, these approaches may overlook mesenchymal CTCs, considered highly malignant. Our aim was to establish a workflow to enrich and isolate patient-matched EpCAMhigh and EpCAMlow/negative CTCs within the same blood samples, and to investigate the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutational status within single CTCs. We sequentially processed metastatic breast cancer (MBC) blood samples via CellSearch® (EpCAM-based) and via Parsortix™ (size-based) systems. After enrichment, cells captured in Parsortix™ cassettes were stained in situ for nuclei, cytokeratins, EpCAM and CD45. Afterwards, sorted cells were isolated via CellCelector™ micromanipulator and their genomes were amplified. Lastly, PIK3CA mutational status was analyzed by combining an amplicon-based approach with Sanger sequencing. In 54% of patients' blood samples both EpCAMhigh and EpCAMlow/negative cells were identified and successfully isolated. High genomic integrity was observed in 8% of amplified genomes of EpCAMlow/negative cells vs. 28% of EpCAMhigh cells suggesting an increased apoptosis in the first CTC-subpopulation. Furthermore, PIK3CA hotspot mutations were detected in both EpCAMhigh and EpCAMlow/negative CTCs. Our workflow is suitable for single CTC analysis, permitting-for the first time-assessment of the heterogeneity of PIK3CA mutational status within patient-matched EpCAMhigh and EpCAMlow/negative CTCs.

The Warwick Agreement on femoroacetabular impingement syndrome (FAI syndrome): an international consensus statement.

The 2016 Warwick Agreement on femoroacetabular impingement (FAI) syndrome was convened to build an international, multidisciplinary consensus on the diagnosis and management of patients with FAI syndrome. 22 panel members and 1 patient from 9 countries and 5 different specialties participated in a 1-day consensus meeting on 29 June 2016. Prior to the meeting, 6 questions were agreed on, and recent relevant systematic reviews and seminal literature were circulated. Panel members gave presentations on the topics of the agreed questions at Sports Hip 2016, an open meeting held in the UK on 27-29 June. Presentations were followed by open discussion. At the 1-day consensus meeting, panel members developed statements in response to each question through open discussion; members then scored their level of agreement with each response on a scale of 0-10. Substantial agreement (range 9.5-10) was reached for each of the 6 consensus questions, and the associated terminology was agreed on. The term 'femoroacetabular impingement syndrome' was introduced to reflect the central role of patients' symptoms in the disorder. To reach a diagnosis, patients should have appropriate symptoms, positive clinical signs and imaging findings. Suitable treatments are conservative care, rehabilitation, and arthroscopic or open surgery. Current understanding of prognosis and topics for future research were discussed. The 2016 Warwick Agreement on FAI syndrome is an international multidisciplinary agreement on the diagnosis, treatment principles and key terminology relating to FAI syndrome.Author note The Warwick Agreement on femoroacetabular impingement syndrome has been endorsed by the following 25 clinical societies: American Medical Society for Sports Medicine (AMSSM), Association of Chartered Physiotherapists in Sports and Exercise Medicine (ACPSEM), Australasian College of Sports and Exercise Physicians (ACSEP), Austian Sports Physiotherapists, British Association of Sports and Exercise Medicine (BASEM), British Association of Sport Rehabilitators and Trainers (BASRaT), Canadian Academy of Sport and Exercise Medicine (CASEM), Danish Society of Sports Physical Therapy (DSSF), European College of Sports and Exercise Physicians (ECOSEP), European Society of Sports Traumatology, Knee Surgery and Arthroscopy (ESSKA), Finnish Sports Physiotherapist Association (SUFT), German-Austrian-Swiss Society for Orthopaedic Traumatologic Sports Medicine (GOTS), International Federation of Sports Physical Therapy (IFSPT), International Society for Hip Arthroscopy (ISHA), Groupo di Interesse Specialistico dell'A.I.F.I., Norwegian Association of Sports Medicine and Physical Activity (NIMF), Norwegian Sports Physiotherapy Association (FFI), Society of Sports Therapists (SST), South African Sports Medicine Association (SASMA), Sports Medicine Australia (SMA), Sports Doctors Australia (SDrA), Sports Physiotherapy New Zealand (SPNZ), Swedish Society of Exercise and Sports Medicine (SFAIM), Swiss Society of Sports Medicine (SGMS/SGSM), Swiss Sports Physiotherapy Association (SSPA).

Free vibrations of a cantilevered SWCNT with distributed mass in the presence of nonlocal effect.

The Hamilton principle is applied to deduce the free vibration frequencies of a cantilever single-walled carbon nanotube (SWCNT) in the presence of an added mass, which can be distributed along an arbitrary part of the span. The nonlocal elasticity theory by Eringen has been employed, in order to take into account the nanoscale effects. An exact formulation leads to the equations of motion, which can be solved to give the frequencies and the corresponding vibration modes. Moreover, two approximate semianalytical methods are also illustrated, which can provide quick parametric relationships. From a more practical point of view, the problem of detecting the mass of the attached particle has been solved by calculating the relative frequency shift due to the presence of the added mass: from it, the mass value can be easily deduced. The paper ends with some numerical examples, in which the nonlocal effects are thoroughly investigated.

Amyloid beta a4 precursor protein-binding family B member 1 (FE65) interactomics revealed synaptic vesicle glycoprotein 2A (SV2A) and sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) as new binding proteins in the human brain.

FE65 is a cytosolic adapter protein and an important binding partner of amyloid precursor protein. Dependent on Thr668 phosphorylation in amyloid precursor protein, which influences amyloidogenic amyloid precursor protein processing, FE65 undergoes nuclear translocation, thereby transmitting a signal from the cell membrane to the nucleus. As this translocation may be relevant in Alzheimer disease, and as FE65 consists of three protein-protein interaction domains able to bind and affect a variety of other proteins and downstream signaling pathways, the identification of the FE65 interactome is of central interest in Alzheimer disease research. In this study, we identified 121 proteins as new potential FE65 interacting proteins in a pulldown/mass spectrometry approach using human post-mortem brain samples as protein pools for recombinantly expressed FE65. Co-immunoprecipitation assays further validated the interaction of FE65 with the candidates SV2A and SERCA2. In parallel, we investigated the whole cell proteome of primary hippocampal neurons from FE65/FE65L1 double knockout mice. Notably, the validated FE65 binding proteins were also found to be differentially abundant in neurons derived from the FE65 knockout mice relative to wild-type control neurons. SERCA2 is an important player in cellular calcium homeostasis, which was found to be up-regulated in double knockout neurons. Indeed, knock-down of FE65 in HEK293T cells also evoked an elevated sensitivity to thapsigargin, a stressor specifically targeting the activity of SERCA2. Thus, our results suggest that FE65 is involved in the regulation of intracellular calcium homeostasis. Whereas transfection of FE65 alone caused a typical dot-like phenotype in the nucleus, co-transfection of SV2A significantly reduced the percentage of FE65 dot-positive cells, pointing to a possible role for SV2A in the modulation of FE65 intracellular targeting. Given that SV2A has a signaling function at the presynapse, its effect on FE65 intracellular localization suggests that the SV2A/FE65 interaction might play a role in synaptic signal transduction.

IR spectroscopy applied subsequent to a proton transfer reaction in the excited state of isolated 3-hydroxyflavone and 2-(2-naphthyl)-3-hydroxychromone.

IR/R2PI-spectroscopy has been applied to the electronic ground and electronically excited states of 3-hydroxyflavone (3-HF) and 2-(2-naphthyl)-3-hydroxychromone (2-NHC) in a supersonic jet yielding direct structural information on the educt and product of a proton transfer reaction. We show that IR spectra of the electronically excited states can be recorded subsequent to a photoinduced chemical reaction, in this case a proton transfer. In combination with DFT and TDDFT calculations structural assignments are performed.

Detection of charge movements in ion pumps by a family of styryl dyes.

A family of fluorescent styryl dyes was synthesized to apply them as probes that monitor the ion-translocating activity of the Na,K-ATPase and the SR Ca-ATPase, similar to the widely used dye RH421. All dyes had the same chromophore but they differed in the length of the spacer between chromophore and polar head, an isothiocyanate group, and in the lengths of the two identical acyl chains, which form the tail of the dye molecules. A number of substrate-dependent partial reactions of both P-type ATPases affected the fluorescence intensity, and the magnitude of the fluorescence changes was used to characterize the usefulness of the dyes for further application. The experimental results indicate that electrochromy is the major mechanism of these dyes. While in the case of the Na,K-ATPase a single dye, 5QITC, showed larger fluorescence changes than all others, in the case of the SR Ca-ATPase all dyes tested were almost equal in their fluorescence responses. This prominent difference is interpreted as a hint that the position of the ion binding sites in both ion pumps may differ significantly despite their otherwise closely related structural features. Quench experiments with spin-labeled lipids in various positions of their fatty acids were used to gain information on the depth of the chromophore of the different dyes within the membrane dielectric, however, the spatial resolution was so poor that only qualitative information on the position of the chromophore in the lipid phase could be obtained.

Effects of acyclo-retinoic acid and lycopene on activation of the retinoic acid receptor and proliferation of mammary cancer cells.

The biochemical mechanisms underlying the inhibitory effects of lycopene, the main tomato carotenoid, on the growth of cancer cells are largely unknown. It has been hypothesized that lycopene derivatives may act as ligands for a nuclear receptor in analogy to retinoic acid, the hormone derived from beta-carotene. The inhibition of human mammary cancer (MCF-7) cell growth and the transactivation of the retinoic acid receptor (RAR) reporter gene by synthetic acyclo-retinoic acid, the open chain analog of retinoic acid, was compared to the effects of lycopene and retinoic acid in the same systems. Acyclo-retinoic acid activated the DR-5 retinoic acid response element with a approximately 100-fold lower potency than retinoic acid. This effect was independent of cotransfection with the RARalpha receptor. Lycopene exhibited only very modest activity in this system. In contrast to the results from the transactivation studies, acyclo-retinoic acid, retinoic acid, and lycopene inhibited cell growth with a similar potency. Preincubation with each of the three compounds slowed down cell cycle progression from G1 to S phase. In summary, acyclo-retinoic acid inhibited cancer cell growth and interacted with RAR. However, it exhibited low affinity for RAR and a correspondingly low efficacy in activating this receptor, indicating that RAR does not mediate the growth inhibitory effect of the compound. In addition, the concentrations of acyclo-retinoic acid and of lycopene required for inducing inhibition of cell growth were similar, suggesting that acyclo-retinoic acid is unlikely to be the active metabolite of lycopene.

Stimulation of gap junctional communication: comparison of acyclo-retinoic acid and lycopene.

Carotenoids and retinoids stimulate gap junctional communication (GJC), thought to be related to cancer-preventive properties. Lycopene, a nonprovitamin A carotenoid and its possible oxidation product, acyclo-retinoic acid, were tested for their effect on GJC, on stabilization of connexin43 mRNA, and on the transactivation of the RAR-beta2-promoter in vitro. In human fetal skin fibroblasts, GJC was stimulated by lycopene and acyclo-retinoic acid. Lycopene was effective at a concentration of 0.1 microM, whereas higher amounts of acyclo-retinoic acid (1 microM) were needed for comparable stimulation. Stabilizing effects of acyclo-retinoic acid on the mRNA of connexin43 via elements located in the 3'-UTR were weak. In comparison to retinoic acid (0.1 microM), considerably higher concentrations of the acyclo analog (50 microM) were required for similar effects; lycopene (0.1 microM) was not active in this system. Likewise, unphysiologically high levels of acyclo-retinoic acid (50 microM) were necessary to transactivate the RAR-beta2 promoter. The data demonstrate that acyclo-retinoic acid is much less active than retinoic acid with respect to GJC and retinoid-related signaling. Therefore, we conclude that lycopene affects GJC independent of the formation of acyclo-retinoic acid.

Biological activities of Apo-canthaxanthinoic acids related to gap junctional communication.

Apo-carotenoids with different numbers of conjugated double bonds are formed upon eccentric cleavage of carotenoids. These compounds may exhibit biological activities similar to those of the parent carotenoids or their central cleavage products, the retinoids. 11-Apo-canthaxanthin-11-oic acid, 13-apo-canthaxanthin-13-oic acid, and 14'-apo-canthaxanthin-14'-oic acid, carrying 2, 3, or 5 conjugated double bonds in the polyene chain, respectively, were tested for their effects on gap junctional communication (GJC), on stabilization of connexin43 mRNA, and on the activation of the retinoic acid-beta2 receptor (RAR-beta2 receptor); the effects were compared to those of retinoic acid and 4-oxo-retinoic acid, known to stimulate GJC and to activate the RAR-beta2 receptor. The effects of 4-oxo-retinoic acid were comparable to those of retinoic acid. 4-Oxo-retinoic acid, like retinoic acid, influences the stability of connexin 43 mRNA via elements located in the 3'-UTR. No effects were observed with the short-chain apo-canthaxanthinoic acids. A small but statistically significant induction of GJC and transactivation activity towards the RARbeta2 was found with 14'-apo-canthaxanthin-14'-oic acid. This might be due to biological effects of the compound itself or to biologically active breakdown products. The data suggest that the major biological effects of canthaxanthin on retinoid signaling pathways are related to activities mediated by the products of the central cleavage.

Biological activities of natural and synthetic carotenoids: induction of gap junctional communication and singlet oxygen quenching.

Induction of gap junctional communication (GJC) and antioxidant activities of carotenoids have been considered as biochemical mechanisms underlying the cancer-preventive properties of these compounds. beta-Carotene and other carotenoids, including those lacking provitamin A activity, proved to be active in both these parameters. The beta-carotene analogs retrodehydro-beta-carotene, echinenone, cryptoxanthin (3-hydroxy-beta-carotene), 4-hydroxy-beta-carotene and canthaxanthin stimulate GJC and efficiently deactivate singlet molecular oxygen. beta-Carotene is less active than its retro-dehydro analog with respect to (1)O2-quenching but GJC is similar. The five-membered ring analog of canthaxanthin, dinor-canthaxanthin, has less effect on GJC as compared with the parent compound but exhibits increased singlet oxygen quenching. Straight-chain polyene dialdehydes are quenchers of singlet oxygen, the efficiency increasing with the number of conjugated double bonds. However, none of these compounds significantly induce GJC. These data indicate that the two properties of carotenoids addressed in this study may operate independent of each other.

Plasma concentrations of carotenoids and tocopherols in male long-term tobacco chewers, smokers and nonusers.

Plasma carotenoid and tocopherol concentrations of men, aged 25 to 55 years, who were long-term chewers, smokers, or tobacco nonusers were determined. Tobacco users had either chewed (n = 11) or smoked (n = 23) for > 15 years. Nonusers (n = 10) had never smoked > 1 pack or chewed > 34 g. Food energy, mono- and poly-unsaturated and saturated fats, cholesterol, vitamin A, vitamin E, and carotenoid intakes of the three groups were not significantly different. Chewers and smokers reported consuming significantly less cryptoxanthin, found primarily in some fruits, and had significantly lower plasma cryptoxanthin levels than nonusers. Nonusers had significantly higher concentrations of plasma alpha-tocopherol than smokers; whereas those of chewers were intermediate. Nonusers had significantly higher concentrations of plasma gamma-tocopherol and total tocopherols than chewers or smokers. Plasma delta-tocopherol concentrations of the groups were not significantly different. Nonusers had significantly higher levels of beta-carotene than smokers but not chewers. Plasma lutein and lycopene concentrations of all groups were not significantly different. Dietary intakes of total carotenoids and tocopherols of the three groups were not significantly different, yet nonusers had higher plasma concentrations of total and most individual carotenoids and tocopherols than smokers with values for chewers being intermediate.

Evaluation of serum protein separation by capillary electrophoresis: prospective analysis of 1000 specimens.

To critically assess the method of capillary electrophoresis (CE) we examined 1000 prospective serum samples submitted for protein electrophoresis by both high-resolution agarose gel electrophoresis (HRAGE) and CE. CE was performed using a 72 cm (50 cm to detector) x 50 microns I.D. fused-silica capillary with detection of absorbance at 200 nm. The 1000 samples examined contained 362 monoclonal paraproteins with concentrations ranging from 1 to 71 g/l. We evaluated the individual paraprotein correlations, the overall correlation between the two methods being 0.96. We found that HRAGE gave slightly higher values for the monoclonal bands than CE and the difference was statistically significant. We conclude that CE is a viable alternative to HRAGE for the determination of protein dyscrasias in a routine clinical laboratory.

Erythrocyte and plasma B-6 vitamer concentrations of long-term tobacco smokers, chewers, and nonusers.

Erythrocyte and plasma B-6 vitamer concentrations were determined in males aged 25-55 y who were long-term smokers, chewers, or nonusers. Tobacco users had either smoked (n = 23) or chewed (n = 11) for > 15 y; nonusers (n = 11) had never smoked or chewed. All subjects had normal hematocrit values. Food energy, protein, and vitamin B-6 intakes of the three groups of subjects were not significantly different. All subjects had fasting plasma pyridoxal-5'-phosphate (PLP) concentrations indicative of adequacy. Erythrocyte B-6 vitamer and 4-pyridoxic acid (4-PA) concentrations of all three groups were not significantly different. Nonusers had significantly higher plasma PLP concentrations than did smokers, whereas PLP concentrations of chewers were intermediate between the two groups. Chewers had significantly higher concentrations of plasma pyridoxamine-5'-phosphate (PMP) than other groups. Plasma pyridoxal, pyridoxine, pyridoxamine, and 4-PA concentrations of the three groups were not significantly different. Differences in some B-6 vitamer concentrations in plasma but not in erythrocytes were observed between tobacco users and nonusers.