The neglected issue of pyridoxal- 5′ phosphate

Mario Plebani; Joris Delanghe

doi:10.1515/cclm-2025-0258

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The neglected issue of pyridoxal- 5′ phosphate

Mario Plebani und Joris Delanghe

Veröffentlicht/Copyright: 21. März 2025

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Aus der Zeitschrift Clinical Chemistry and Laboratory Medicine (CCLM) Band 63 Heft 6

Can you tell us your story on PLP?

Mario Plebani: My medical degree thesis in July 1975 was entitled ‘Study of the influence of pyridoxal phosphate on the determination of transaminase activities’. The data obtained emphasized the importance of adding pyridoxal-5′-phosphate to the reagents used for the determination of alanine- (ALT) and aspartate-aminotransferase (AST) to optimize the results, particularly in the case of patients with vitamin B6 deficiency/diminution. These results prompted me to plan further studies that concluded with my first publication in an international journal on the reference intervals for ALT and AST after addition of pyridoxal-5′-phosphate [1]. 10 years later, the International Federation of Clinical Chemistry, now renamed International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), issued and published the recommendations on methods for the measurement of catalytic concentration of enzymes, namely AST and ALT [2], 3]. We recently have published a paper dealing with the need to add pyridoxal-5′-phosphate (PLP) to the reagents used for the determination of AST and ALT [4). In fact, after 5 decades of my work and 4 decades of the IFCC recommended methods, adherence to the guideline is still poor and many, if not the majority, of clinical laboratories do not add PLP.

Why are the vendors not always following the guidelines

Joris Delanghe: Adding 100 μmol/L concentration of pyridoxal-5′-phosphate to the reaction mixture results in an increase in absorbance at 340 nm of approximately 0.2, which limits the dynamic range of the ALT assay [4], 5]. This reduced the dynamic range of the ALT assay. which could eventually lead to a rerun of the test on a diluted sample, thus increasing the analytical turnaround time (TAT).

Adding PLP to the reagent decreases the reagent stability. Furthermore, omitting PLP results in a systematic reduction of the ALT activity [6]. In extreme situations, false negative ALT results may be observed in case of a vitamin B6 deficiency. PLP deficiency is relatively rare. Knowing that plasma ALT activity is used as a surrogate marker for hepatotropic viruses, a false negative ALT value in a donor could have legal consequences. It is remarkable to see that vendors sell ALT kits in the US which follow the IFCC recommendations whereas in Europe the modified (not containing PP) are on the market, which may be related to the risk being sued.

In case of homocysteinemia, one of the causes is PLP deficiency. In this way the finding of low ALT activities could be the clue for diagnosing hyperhomocysteinemia, an underused cardiovascular risk factor [7].

It should be mentioned that the situation for ALT is not unique [4]: even after the implementation of reference material SRM 967 for serum creatinine, not all instruments have been recalibrated to the new global creatinine standard. The glycerol error in triglyceride assay is widely accepted, and assays which corrected for this error have even been withdrawn from the market and the lack of adherence to currently published guidelines has been already highlighted [8], 9].

Can you estimate the prevalence of PP deficiency in the general population?

Mario Plebani: While overt vitamin B6 deficiency is a rare condition and is mainly defined by the occurrence of specific clinical signs or symptoms, suboptimal vitamin B6 status is observed in many diseases including cardiovascular diseases (CVDs), stroke, deep venous thrombosis, diabetes mellitus, chronic liver diseases and cancer [9], 10]. Vitamin B6 deficiency, therefore, is rather a notion in evolution since novel at-risk pathological conditions seem due to a degree of impairment that does not reach the level of deficit classically defined as a clear vitamin B6-deficient status [11]. As further evidence, in our recently published paper [12], a significant number of randomly selected patient samples were found to have low levels of PLP.

Outlook

Mario Plebani: In my opinion, the fundamental problem is the lack of PLP availability in the reagents provided by many manufacturers, and the complete lack of information in the IFU on the need for its addition. The aforementioned analytical concerns associated with the addition of PLP, including limited on-board stability and the increase in absorbance at 340 nm which limits the dynamic range, can be easily addressed by experienced laboratory staff. However, although in vitro supplementation with PLP is important to optimize the measurement of the catalytic activity of both AST and ALT, based on a sound pathophysiological background (vitamin B6 deficiency), and clinically essential to avoid erroneous results due to vitamin B6 hypovitaminosis, it is not carried out in the real world. It is surprising that the organizers of external quality assurance (EQA) schemes, as well as accreditation bodies, do not sufficiently address this fundamental analytical issue, which has a significant negative impact on the accuracy of results and patient diagnosis. But the true question to ask is, “What would patients say if they knew that, 50 years later, laboratory professionals disregarded recommendations issued by scientific bodies that have important implications for the quality of results and safety of diagnosis”?.

Corresponding author: Prof. Dr. Mario Plebani, Department of Medicine-DIMED, University of Padova, Padova, Italy, E-mail: mario.plebani@unipd.it

This editorial is published simultaneously in Clinical Chemistry and Laboratory Medicine (CCLM, DOI: https://doi.org/10.1515/cclm-2025-0258, and Clinica Chimica Acta (CCA), DOI: 10.1016/j.cca.2025.120232. The manuscript was originally submitted to CCA.

Research ethics: Not applicable.
Informed consent: Not applicable.
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
Use of Large Language Models, AI and Machine Learning Tools: None declared.
Conflict of interest: The authors state no conflict of interest.
Research funding: None declared.
Data availability: Not applicable.

References

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2. International Federation of Clinical Chemistry (IFCC), Hørder, M, Rej, R. Approved Recommendation (1985) On IFCC methods for the measurement of catalytic concentration of enzymes. IFCC method for aspartate aminotransferase. J Clin Chem Clin Biochem 1986;24:497–510.Suche in Google Scholar

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12. Talli, I, Marchioro, L, Zaninotto, M, Cosma, C, Pangrazzi, E, Artusi, C, et al.. Measurement of AST and ALT with Pyridoxal-5′-Phosphate according to IFCC: a decades-long gap seems to be filled. Clin Chim Acta 2025;569:120158. https://doi.org/10.1016/j.cca.2025.120158.Suche in Google Scholar PubMed

Published Online: 2025-03-21

Published in Print: 2025-05-26

Artikel in diesem Heft

https://doi.org/10.1515/cclm-2025-0258