ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 16, No. 2 Copyright © 1986, Institute for Clinical Science, Inc. Review of Pyridoxal Phosphate and the Transaminases in Liver Disease RAYMOND E. VANDERLINDE, Ph.D. Department of Pathology and Laboratory Medicine, Hahnemann University, Philadelphia, PA 19102 ABSTRACT In vitro supplementation with the active form of vitamin B6, pyridoxal- phosphate (PLP), increases measurements of both serum aminotransferase enzymes, L-aspartate: 2-oxoglutarate amino transferase, EC 2.6.1.1 (AST) and L-alanine: 2-oxoglutarate aminotransferase, EC 2.6.1.2 (ALT). The plasma PLP level in normal individuals clearly relates inversely to the degree of stimulation of serum AST and ALT. PLP added in vitro increases the reference values but does not decrease the biological vari­ ability of AST measurements in healthy individuals. Since B6 deficiency is observed in alcoholics, in some significant per­ centage of hospitalized patients and in apparently healthy people over age 64, these individuals will show PLP stimulation of their serum amino­ transferase enzymes. Patients with liver disease show lesser activation with PLP of AST activ­ ity but not ALT activity than patients with heart disease (myocardial infarction). AST isoenzyme measurements in the form of a mitochondrial AST/total AST ratio may discriminate alcoholic hepatitis from all other hepatic diseases. In renal dialysis patients including transplant patients, it may be desirable to measure the aminotransferases with added PLP in order to reflect better the cytolytic state of the liver. While unconfirmed studies suggest the combination of PLP activation and AST isoenzyme measurements may aid in the diagnosis of hepatoma, PLP activation per se does not provide clear cut improved diagnostic value of AST and ALT in liver diseases. However, in view of PLP incorporation into the IFCC ref­ erence methods for AST and ALT, and the National Reference System for the Clinical Laboratory, it is recommended that PLP be included in all AST and ALT measurements. Introduction bohydrate metabolism. Both amino­ transferases require vitamin B6, pyridox- The enzymes AST and ALT carry ine, at their active sites in the out the interconversion of amino acids physiologically active form of pyridoxal- and alpha-keto acids by the transfer of 5'-phosphate (PLP). Hamfelt20 22 in 1964 amino groups and thus function as a link was the first to demonstrate and we con­ between amino acid metabolism and car- firmed in 1973 the increased AST activ­ 79 0091-7370/86/0300-0079 $02.00 © Institute for Clinical Science, Inc. 80 VANDERLINDE ity of normal and pathological sera after AST Survey.09 In each instance, conver­ in vitro supplementation with PLP . 08 sion brings about convergence of multi­ Subsequently, we demonstrated the rate ple method mean data bars to a single of association of PLP and the apoenzyme coherent mean data bar. was retarded by phosphate buffer in comparison with tris (hydroxymethyl) Relationship of Vitamin B6 Deficiency aminomethane (Tris) or six other organic buffers.60 Numerous investigations of the Vitamin B6 is widely distributed in effect of measurement conditions on the most common foodstuffs, but losses from catalytic activity of both aminotransfer­ food of up to 70 percent can occur during ases have been made over the past ten cooking, processing, and refining. The years, which have been reviewed body’s need for Vitamin B6 appears to be recently by Rej.37 Clearly the serum directly related to protein intake.67 Also aminotransferases are not fully saturated certain clinical situations such as expo­ with the coenzyme. sure to radiation, drug therapies such as While the activation findings for both isoniazid, penicillamine, anovulatory normal and pathological sera are highly agents, etc. and cardiac failure as well as variable, the analytical reasons for these pregnancy and lactation increase the differences have been only partly clari­ requirement.63 The coenzyme forms of fied and the variation may also reflect Vitamin B6 are known to participate in nutritional status. Thus, Westerhuis and more than 50 enzymatic reactions Hafkenscheid77 found a very significant involved in the metabolism of amino inverse linear relationship between the acids. A recent review has addressed plasma PLP concentration and the these and the other functions of PLP in amount of stimulation of AST or ALT in considerable detail.68 healthy individuals. Nevertheless, the The main form of the vitamin in the International Federation of Clinical plasma is PLP which upon hydrolysis Chemistry reference methods for AST3 yields pyridoxal (PL). Human body and ALT6 as well as the American Asso­ stores are minimal and can be depleted ciation for Clinical Chemistry recom­ quickly. While either urinary 4-pyridoxic mended methods00-61 include supple­ acid in 24-hour specimens or the 4-pyri­ mentation with PLP. In addition, several doxic acid per mg of creatinine excretion laboratories, reporting in the CAP Enzy- in random urine can be used to measure mology Survey,42 utilize commercially vitamin B6 status in humans67, the plasma available kit methods incorporating PLP or red cell level of PLP as well as the while many others do not. However, tryptophane load test are good indices of now that Bowers and McComb7 have the nutritional status of B6.21 Thus, introduced the International Clinical Hamfelt20 and Ranke et al33 showed Enzyme Scale (ICES) concept, compari­ marked decreases in PLP level with age son of results by different methods may which diet supplement corrected in be less of a problem. They propose that some but not all individuals. Further­ all AST methods can be systematically more, Hamfelt20 found that persons over converted to the ICES Scale and that all 64 years of age had 30 percent lower AST results will be numerically compati­ levels of serum AST than groups aged ble. Demonstration of its applicability 41-63 or 18-40, and showed a higher has been made by converting the AST percentage stimulation by AST (19 per­ results reported in the 1983 College of cent vs. 6.5 percent vs. 5 percent); American Pathologists’ Comprehensive enzyme activity was not restored to the Chemistry Survey7 and a New York State levels shown by the younger groups. PYRIDOXAL PHOSPHATE AND TRANSAMINASES IN LIVER DISEASE 81 Although the more recent work of catalytically inactive apo-AST in serum Deledda12 tends to support Hamfelt’s were activated by saturating it with pyri- findings and they appear to hold for ALT doxal phosphate. However, Harder and as well, more definitive studies are Bowers24 found the biological variability needed. (CV’s) of serum AST measurements of While an increased intake of pyridox- nine healthy individuals over a two week ine for a few days had only a slight effect period with and without in vitro PLP on the concentration of circulating AST stimulation to be the same. They pre­ apoaminotransferase04 in healthy indi­ sume that their results regarding the bio­ viduals, Vitamin B6 deficiency may con­ logical variabilities of AST measured tribute significantly to interindividual with and without PLP for their selected differences in the PLP stimulation of subset of persons would hold true also serum ALT and AST in hospitalized for a universe of the healthy population. patients.22 In another study39 Leevy et al While they observed an average 39 per­ observed that 27 percent of randomly cent increase in serum AST of normal selected municipal hospital patients had healthy individuals upon the addition of low serum levels of vitamin B6. PLP, a 49 percent increase was observed Very recently, Westerhuis and Haf- for 48 sera from hospitalized subjects all kenscheid77 reported a very significant of whom had AST values within normal inverse relationship between the plasma range. Obviously the incorporation of PLP concentration in normal, healthy PLP alters the reference ranges upward individuals and the stimulation of serum for ALT and AST as shown by Plebani et ALT and AST by in vitro PLP, i.e., the al51 who established reference values for degree of stimulation of the apoenzyme optimized ALT and AST measurements of the two serum aminotransferases supplemented with PLP. clearly depends inversely on the plasma PLP concentration in vivo. Plasma from General Clinical men showed a 30 percent higher mean value than plasma from women but an A very important question is whether age factor was not included. The coeffi­ the addition of PLP leads to greater cient of variation for the ALT determina­ diagnostic utility of the aminotransfer­ tions is about twice as large as for the ases, i.e., better differentiation between AST determinations on the same healthy disease states. individuals, which may reflect a differ­ In 1966 Hamfelt22 measured AST with ence in biological variation. However, and without added pyridoxal in old per­ their most significant observation was sons, in acute intermittent porphyria, in the fact that the degree of in vitro stim­ heart muscle necrosis, and in thyrotoxi­ ulation of both serum AST and ALT in cosis. In addition, he measured the healthy normal individuals is inversely effect of pyridoxal on both aminotrans­ proportional to the in vivo plasma level ferases in patients with chronic alcohol­ of PLP. While the authors speculated ism. Significant changes were observed about the underlying cause in terms of in all groups of patients except for three sex, aging of cells, etc., no valid explana­ of six measurements of ALT in chronic tion was arrived at. alcoholic patients. Later Hamfelt and Does the addition of PLP decrease the Tuvemo23 studied the effect of exoge­ biological variability of the enzyme mea­ nous vitamin B6 on the maternal serum surements? Rej and Vanderlinde58 pos­ and red cell AST activities at delivery, tulated that the dispersion of reference assayed in the presence and absence of values of AST might decrease if all of the PLP, as well as the cord blood activity.