Review of Pyridoxal Phosphate and the Transaminases in Liver Disease RAYMOND E

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. 82 VANDERLINDE Many groups during the middle nine of Raitke and Moss54 and Rosalki and teen seventies including Ury and Bayoumi.62 Chassy,73 Cheung and Briggs,10 Rosalki As part of the IFCC recommended and Bayoumi,62 Moss,47 Ratnaike and procedure for serum AST,5 as modified Moss,54 Jung and Egger,26 and Jung and for use with a centrifugal analyzer, Bohm23 studied the effect of in vitro PLP Bruns, Savory et al8 in 1981 evaluated supplementation on aminotransferase the effect of PLP on serum AST from assays in various diseases and conditions. four types of patients: liver, heart, pedi These data, based on various assay sys atric and renal. Sera from patients with tems for AST and ALT, were reviewed heart disease (myocardial infarction) by Campbell and Sanderson9 in 1979. In showed more stimulation by PLP than general, they suggest no clear cut those from any of the other three groups. increased diagnostic utility perhaps ow In 1983 Dols and van Zanten15 published ing to the highly variable factors affecting their findings comparing the German the extent of aminotransferase activation Society for Clinical Chemistry (DGKC) with PLP including: (1) concentration of recommended AST method, the first PLP, (2) duration of preincubation, (3) “optimized” procedure which is carried the buffer system used since phosphate out in phosphate buffer and does not inhibits60 the reassociation of PLP and utilize PLP, with the IFCC method3 apoenzyme, (4) concentration of amino which utilizes PLP and tris buffer. The transferase substrates, and (5) patient indices of intra- vs. interindividual vari variability. However, significant stimula ability showed that reference ranges by tion was observed in a number of disease both methods are insensitive to intra categories including myocardial infarc individual variations in serum AST activ tion (MI), liver diseases, conditions ities, in agreement with results pub requiring extracorporeal blood treat lished by Harder and Bowers.24 Thirty of ment, nutritional deficiency and preg 745 results for patients’ samples were nancy which demonstrated the need for identified as abnormal by the IFCC- the better controlled studies which have based method but normal by the DGKC evolved more recently. method; 13 were classified as normal by Bergmeyer, Scheibe and Wahlefeld4 the IFCC-based method but abnormal in 1978 developed optimized methods by the DGKC method. for both serum AST and ALT and as part The patients’ samples were subdivided of their study described the effects of into the categories of kidney, liver, heart PLP on pooled sera from either patients and unclassified. The correlation with diagnosed heart (myocardial infarc between AST values by each method for tion) or liver disease. Their careful stud samples obtained from heart patients ies included individual sample blanks. was poorer than for the other patient cat Both serum AST and ALT from liver dis egories. This phenomenon apparently is ease patients showed about 15 percent produced by the large spread in PLP stimulation by PLP. On the other hand, activation for samples from patients with serum AST from heart disease patients heart disease as has been previously showed a 49 percent stimulation whereas observed by others not using IFCC- serum ALT showed only 7.3 percent. based methodology.54,62 Thus PLP stim Thus, sera for AST from patients with ulation of samples from patients with heart disease show greater activation heart disease is more variable as well as with PLP than sera for AST or ALT acti larger than observed for patients with vation from patients with liver diseases. kidney, liver or unclassified diseases. These results confirm the earlier findings However, Dols and van Zanten’s stud PYRIDOXAL PHOSPHATE AND TRANSAMINASES IN LIVER DISEASE 83 ies15 demonstrated that AST/ALT ratios mean PLP stimulated AST level from obtained by the IFCC methods with 118 ± 17 to 146 ± 20 U/l which is not PLP added, carried out on sera from 101 statistically significant. Secondly, plasma patients suffering from biopsy-proven PLP tends to be reduced in patients with liver disease, were still utilizable. In a extrahepatic obstruction.45 This may be comparative study of improved DuPont due to the hydrolysis of plasma PLP by ACA methodology with added PLP in alkaline phosphatase in conditions with 1981, Garber, Feldbruegge, and Hoes- raised alkaline phosphatase.3 Thirdly, sel18 found highly variable percentages of the plasma clearance of 31 ml/min for stimulation by PLP in a small number of PLP in healthy individuals is increased patients with MI, on dialysis with carci in several types of liver disease:45 noma of the colon with hepatic involve namely, alcoholic cirrhosis—63 ml/min ment, with hepatoma, or metastatic mel (2x normal); acute viral hepatitis—106 anoma with liver involvement. The ml/min (3x normal) and in extrahepatic patients with various types of malignant obstruction—132 ml/min (4x normal). liver pathology showed a greater These increases in the plasma clearance increase in observed AST than reported of PLP are presumably due to the earlier by Rosalki and Bayoumi62 for increased removal of plasma PLP by the chronic liver disease. Thus studies “sick” liver, possibly by an alkaline phos involving correlation of various general phatase located in the hepatocyte plasma ized categories of pathology with PLP membrane. On the other hand, Roussow stimulation of the aminotransferases et al64 found that in fulminant hepatic have shown higher results but no clear failure the plasma level of PLP changed cut improved diagnostic utility. markedly, increasing within one week of the onset of symptoms from a mean of Liver 11.3 to 79.8 ng/ml. In parallel with the rise in PLP, there was an increase in A number of investigations involving serum AST holoenzyme activity. PLP activation of the aminotransferases An additional complexity to keep in have been directed to the study of mind is that while ALT is a single soluble patients with specific liver diseases. cytoplasmic enzyme, AST activity con In table I are summarized the effect of sists of a soluble or cytoplasmic enzyme PLP on aminotransferase assays in var (c-AST) and in addition a mitochondrial ious liver diseases and other conditions isoenzyme (m-AST), each of which can to be described. be stimulated differently by PLP and is In evaluating the effects of in vitro best measured with different substrate PLP supplementation on serum AST and concentrations of alpha-ketoglutarate. As ALT in patients with liver disease, sev an example, the AACC recommended eral observations should be kept in method61 for total AST (t-AST) utilizes a mind. According to Mitchell et al,4° base higher alpha-ketoglutarate concentration line plasma PLP concentrations are sig than does the IFCC recommended nificantly lower in cirrhotic patients than method0 for total AST and would there in normal persons owing to the abnormal fore be expected to measure more opti regulation of plasma PLP by cirrhotics as mally m-AST when both isoenzymes are measured by a test dose of PLP. Rous- present in the serum. sow et al63 who observed that approxi Recently, Rej56 has shown that catalyt- mately 90 percent of patients with severe ical measurements and immunological cirrhosis are vitamin B6 deficient, found measurements give about equivalent that treatment with B6 increased the values for the two isoenzymes of AST TABLE I 00 Effect of Pyridoxal Phosphate on Aminotransferase Assays in Various Liver Diseases and Other Conditions "'"

Reference Diseases or Conditions Studied Significant Findings

Horder and Bowers (1977)22 Healthy persons Biological variability of AST measurements is the same with or without in vitro PLP supplementation. Westerhuis and Hafkenscheid Healthy individuals Plasma PLP levels are inversely proportional to AST/ALT (1983)6 stimulation. Bergmeyer, Scheife, and Myocardial infarction (MI) Sera for AST from patients with heart disease (MI) Wahlefel~ (1978)32 Liver diseases showed 3X greater activation with PLP than AST and ALT activation from patients with liver disease. Bruns, Savory et al (1981)33 Liver disease Sera from patients with heart disease showed more Heart disease (MI) stimulation of AST by PLP than patients in other three Pediatric patients under 12 groups. Renal disease Dols and van zanten (1983)34 Heart disease Sera from patients with heart disease showed markedly ;; Liver disease different activation with PLP as compared with sera Z Chronic renal disease from other patients categories. o Miscellaneous disease t<1 t" Dols and van Zanten (1983)34 Heart disease Ratio for AST/ALT, as used in the differentiation of Z" Liver disease liver disease, can be used either with or without PLP o Chronic renal disease activation. t<1 Miscellaneous disease ROussouw, Labadarious et al Cirrhotic patients In vivo treatment with PLP increased the mean in vitro (1978)37 PLP stimulated AST level from 118 to 146 U/l (not significant) . Jung, Ohlrich et al (1978)42 Health persons Serum AST is activated by 37% and ALT by 15% for Chronic liver disease (7 healthy persons. subtypes) Apo AST activity in patients with chronic liver Acute viral hepatitis diseases is not changed as compared with that of healthy personp. However, the relative stimulation rate is significantly smaller. Apo ALT activity and corresponding relative stimulation is significantly greater as compared with healthy persons. In acute viral hepatitis, a decrease of AST and ALT activity is followed by a decrease in the PLP stimulation (less apoenzyme) in the course of the disease. In the authors' experience, the diagnostic value of determination of the aminotransferases could not be improved by the addition of PLP. TABLE I I continued!

Effect of Pyridoxal Phosphate on Aminotransferase Assays in Various Liver Diseases and Other Conditions

Reference Diseases or Conditions Studied Significant Findings

Matloff, Selinger, and Kaplan Healthy individuals The AST/ALT ratio in serum and liver tissue was (1980)h7 Alcoholic liver disease increased only in individuals with alcoholic liver Primary biliary cirrhosis disease but did not reach statistical significance. DISEASE LIVER IN TRANSAMINASES AND PHOSPHATE PYRIDOXAL The addition of saturating amounts of PLP to the Karmen assays did not increase ALT or AST activity of liver tissue of patients with alcoholic liver disease and chronic active hepatitis. Diehl, Potter et al (1984) 49 Alcoholic hepatitis Addition of PLP to liver homogenates increased liver ALT but not AST, in all patients with initially low plasma PLP. Karnei, Ohkubo, and Yamanaka Healthy individuals Significantly higher ratios of apo (PLP stimulation) to (1979) 51* Hepatoma holo AST activity for both the serum mito and Acute hepatitis cytoplasmic AST isoenzymes were observed in patients Miscellaneous liver disease with hepatoma than in patients with acute hepatitis (6 pts.) and in patients with miscellaneous liver diseases (5 pts.) Campbell and Sanderson (1979)31 Dialysis patients PLP produced a stimulation of 29-136% in serum AST from 7 to 9 dialysis patients and of 64-258% in serum ALT with all 9 dialysis patients showing increases. Lacour et al (1982, 1983)68r69 Patients with kidney PLP supplementation resulted in 56% higher AST and 71% transplant higher ALT in patients with kidney transplants in the absence of chronic renal failure. Jung, Ohlrich et al (1981)7* Patients with kidney PLP must be added to the reaction mixture if one is to transplant determine the aminotransferases, especially ALT, correctly in patients with kidney transplants and thus to diagnose liver diseases in such patients. Jung, Mildner et al (1981) Chronic hemodialysis patients The corresponding relative stimulation rates of serum Kidney transplant patients AST and ALT by PLP were not changed in the hemodialysis patients but in renal transplant recipients the relative stimulation of AST was significantly smaller and that of serum ALT was greater. Therefore, PLP addition to the reaction mixture for determination of ALT activities in patients with renal allografts should be used for liver assessment in these patients. Hafkenscheid, Rosier and Patients with renal allografts While serum AST stimulation was the same for patients van Dijk (1984)73 with renal allografts as for healthy individuals, the oo stimulation of ALT was greatly increased except for w patients who received cyclosporine A instead of azathioprine. 86 VANDERLINDE present in liver tissue. However, he patients in the groups limit the conclu demonstrated that the catalytic methods sions which are available. For healthy which we currently use to measure the persons there was significant correlation AST isoenzymes in plasma grossly between the value of either apo AST or underestimate the mitochondrial pro relative stimulation and m-AST, but this portion of the total AST activity and the correlation was lost in liver disease con amount of plasma enzyme present by firming the earlier findings of Rosalki catalytic measurements to be far less and Bayoumi62 and Ratnaike and Moss.54 than found by immunological assay. On In the case of acute viral hepatitis, a the other hand, Leung et al41 found that decrease of AST and ALT activity is fol the proportion of catalytically active lowed by a decrease of ALT apoenzyme c-AST found in serum samples to be activity in the course of the disease. about one-half that of the available isoen However, in their overall experience the zyme, whereas nearly all of the m-AST diagnostic value of determinations of was catalytically active. Thus, in either AST and ALT aminotransferase activities event, current methodologies for serum in liver disease could not be improved AST may fail to measure a major portion by addition of PLP. of the t-AST enzyme actually present, Miyake46 studied the release of the presumably because it is not enzymati AST isoenzymes and ALT in 43 healthy cally active. Immunologic mass unit (ng/ controls and in 280 patients with liver ml) measurement of the AST isoenzymes diseases. High m-AST activity was found as well as total AST and total ALT may in the acute stage of acute hepatitis, in one day provide us with data offering subacute hepatitis and in primary biliary many new insights into the pathophysiol cirrhosis in which conditions were gener ogy of liver disease. ally associated with high total AST activi Let us now consider currently avail ties. The activity ratio of m-AST/t-AST able published investigations on the role varied depending on the stage and sever of PLP activation of AST and ALT in ity of the liver disease. The m-AST/t- liver diseases. Jung et al29 investigated AST ratio was decreased in acute, ful the amounts of c-AST, m-AST, apo and minant and subacute hepatitides while total AST and apo and total ALT in the no significant reduction in ratio was serum of healthy individuals as well as found in bile duct obstruction, alcoholic patients having any of seven types of liver injury, or metastatic liver cancer. chronic liver disease. They found the apo Although relatively high m-AST/t-AST AST activity in patients with various ratios were found in some patients with chronic liver diseases to approximate severe hepatic injury, Miyake found no those found in healthy persons even definite association with a poor prog though the latter had increased amounts nosis. Also observed was a marked ele of total AST activity. Thus, patients with vation of t-AST over ALT in advanced chronic liver disease showed signifi chronic hepatitis, liver cirrhosis and pri cantly smaller PLP stimulation rates for mary hepatoma perhaps as Miyake sug AST than healthy persons. On the other gests owing to a preferential leakage of hand, apo ALT activity and its corre cytoplasmic AST. Skrha et al70 also sponding relative stimulation was signifi investigated the isoenzymes of AST in cantly greater in patients with chronic chronic liver diseases which included liver disease than in healthy persons. chronic hepatitis in 27 patients, liver cir While there were some partly significant rhosis in 23 patients, and secondary neo differences between the various groups plastic affection of the liver in 6 patients. with liver diseases, the small numbers of All patients with biochemically active PYRIDOXAL PHOSPHATE AND TRANSAMINASES IN LIVER DISEASE 87 forms of liver disease manifested day from patients with alcohol problems. increased t-AST as well as cytoplasmic Hepatic ALT activity was significantly AST and 57% manifested simultaneously decreased in the liver tissue of patients increased m-AST. Skrha and his asso with alcoholic hepatitis and cirrhosis as ciates observed that 13% of the patients compared with the activity in individuals with stabilized forms of liver disease with normal livers and individuals with manifested an isolated increase of the primary biliary cirrhosis. The decreased m-AST which they believe could be of hepatic ALT activity was not related to value in evaluating the course of the dis the presence of the cirrhosis in the ease. Also they observed a strikingly biopsy specimens and was not increased high share of m-AST in focal processes of by the addition of saturating amounts of malignant character in the liver. PLP PLP to the assay mixture. The AST/ALT stimulation tests were not carried out as ratio in serum and liver tissue increased a part of either Miyake’s46 or Skrha et only in individuals with alcoholic liver al’s70 investigations. disease and not in four other types of More recently, Jung and Pergande in chronic liver disease. Since Matloff and association with Rej, Schreiber and his coworkers did not measure the serum Schimmelpfennig30 measured the activi or hepatic vitamin B6 content of their ties of two mitochondrial enzymes AST patients and did not perform liver biop and glutamate dehydrogenase (EC sies in alcoholic patients without liver 1.4.1.2) (GLDH), in the sera of healthy disease, their data do not shed major persons and in 43 patients with chronic light on the role of PLP deficiency in liver diseases. The distribution of activi chronic alcohol abuse. ties for GLDH, but not m-AST, was sex More recently, Kakuma, Leevy, dependent with men showing higher Frank and Baker32 have shown that PLP values. While there was a very weak cor protects in vivo and in vitro against the relation between the activities of the two liver cytotoxicity of acetaldehyde, an mitochondrial enzymes in healthy per hepatotoxic metabolite of ethanol. In a sons (r = 0.439), patients with chronic subsequent 1984 paper, Diehl et al13 liver disease exhibited a greater increase studied the relationship between PLP in the activity of GLDH than of m-AST deficiency and activities of serum and and the correlation between the two liver AST and ALT in 12 patients with mitochondrial enzymes was stronger. alcoholic hepatitis. According to their The diagnostic sensitivity and specificity findings, the patients with lower initial of either mitochondrial enzyme are plasma PLP levels did not have signifi reported to be less than that of total AST, cantly more severe acute hepatocellular ALT or gamma-glutamyltransferase (EC injury histopathologically. Hence, Diehl 2.3.2.2). et al’s data do not support the contention that adequate PLP levels protect the Chronic Alcohol Abuse and PLP liver from alcohol induced cytotoxicity. Neither does PLP deficiency seem to In 1974 Lumeng et al43 suggested a explain the low liver AST levels in possible relationship between chronic patients with alcoholic hepatitis because alcohol abuse, PLP deficiency and low neither in vivo nor in vitro did PLP sup ered levels of hepatic ALT activity. plementation bring about a change in Thus, Matloff, Selinger and Kaplan44 AST levels in the liver. However, PLP in investigated AST and ALT levels with vivo did bring about a decrease in serum and without added PLP in liver biopsy AST and an increase in serum ALT. specimens and in sera obtained the same Hence, PLP depletion appears to be par 88 VANDERLINDE tially responsible for the high serum other liver diseases. There was negligi AST/ALT ratio that is typical of patients ble overlap of the hepatoma group with with alcoholic hepatitis. all other groups. To date there has been Panteghini et al49,50 sought to establish no followup of these intriguing findings a relationship between the AST isoen reported in 1979; perhaps owing to the zyme levels in serum and the degree of technical availability and complexity of hepatic damage in a group of 69 patients the assays combined with the greater with various hepatic diseases. During rarity of hepatoma in the American pop hepatic damage, c-AST was found in ulation. However, an immunoprécipita greater quantities than m-AST but the tion assay, using antiporcine antibody latter increased to a greater extent in conjugated to sheep erythrocytes, has alcoholic hepatitis. Thus, the most signif been described74 and is the basis for a icant new data were the much greater diagnostic “kit” method from Eiken increase in plasma m-AST and the ability Chemical Co. Ltd. in Japan, and now of the m-AST/total AST ratio to discrim available in North America.56 inate between alcoholic hepatitis and all other hepatic diseases. PLP was not Reye’s Syndrome added to any of the assays in their study which emphasized the role of the AST Similarities between Reye’s syndrome isoenzymes. The role of m-AST in esti and fulminant hepatic failure suggest mating liver cell structural damage has that these illnesses may share many bio been previously emphasized by Skrha et chemical features. Thus Faraj et al17 al70 and the condition has been showed the concentrations of PLP in described as a “necrotic type” by plasma are significantly higher at the Schmidt and Schmidt.66 onset of disease than after treatment. Recently, Evans et al16 have reported Further, the concentrations of PLP in that serum AST and GGT measurements plasma at the time the patients entered in alcoholics admitted to a detoxification the hospital correlated at the 0.73 level unit are not valid indices of alcohol with their ALT activities and suggests intake since the correlations were rather that the two may be released together low and the sensitivity was only about 50 from the liver. The low plasma concen percent. trations of PLP observed after treatment presumably reflect the reversible pro Hepatoma cess of hepatic injury and also may rep resent decreased hepatic stores of PLP The most striking study reported to in Reye’s patients. date is by Kamei et al33 who combined immunological separation of the AST iso Blood Banking enzymes with their stimulation by PLP in patients with a variety of hepatic dis Although not involving PLP stimula eases. Significantly higher ratios of apo tion, three major prospective studies to holo AST activity for both the mito have established a correlation of elevated and soluble isoenzymes were observed levels of ALT in donor blood with in the patients with hepatoma in contrast increased risk of non-A, non-B hepatitis with cirrhosis, acute hepatitis, chronic (NANB) in blood recipients.1,2’31 Thus hepatitis and other hepatic disorders. directors of donor programs such as the Thus patients with hepatoma differed Greater New York Blood Program have significantly in the amount of PLP stimu instituted ALT testing of all donor lation observed from patients with all bloods, while other experts question PYRIDOXAL PHOSPHATE AND TRANSAMINASES IN LIVER DISEASE 89 whether such enzymatic screening of dialyzed uremic patients, and note loss of donor bloods should be done.52 While PLP does not occur in the plasma ultra answers are not available at this time on filtrates as a result of the hemodialysis. A this important issue, Kolins35 has shown similar PLP deficiency was observed in recently that although the predicative kidney transplant patients with normal value of a negative ALT test is 95 per or near normal renal function, that is, in cent, the predicative value of a positive the absence of chronic renal failure. ALT test is only 30 percent. This means They believe that a chronic deficiency in only 30 percent of the samples with ele tissue PLP concentration induces the vated ALT levels will cause non-A, decrease in apo-transaminase synthesis, non-B post transfusion hepatitis, the which is observed generally in hemodial remaining 70 percent being false posi ysis patients in the absence of cytolytic tives. Kolins feels this low predicative liver disease.72,75,78 Major metabolic value has prevented acceptance of the effects through in vivo repletion with B6 test. On the other hand, Silverstein et have been demonstrated in maintenance al69 through a cost-effectiveness analysis dialysis patients by Kleiner et al.34 found that sensitivity analyses indicate Following up on Wolf’s78 and War- that screening would be cost-saving for a nock’s75 findings of low AST activity in wide range of cost estimates and number hemodialysis patients, Campbell and of units per transfusion. They concluded Sanderson9 investigated the effect of in that ALT screening is warranted until vitro PLP supplementation on amino more sensitive and specific screening transferase assays of serum from dialysis tests for transmissibility of non-A, non-B patients. They obtained an increase hepatitis become available. stimulation of 29-136 percent in serum AST from 7 of 9 dialysis patients and an Renal even greater stimulation of 64-258 per cent in serum ALT with all 9 dialysis In 1972 Wolf and his colleagues78 patients showing increases. The extent of observed, as a clinical laboratory finding, activation in several of the samples was low AST activity in the sera of patients sufficient to cause the observed enzyme undergoing chronic hemodialysis. level to increase from a normal or bor Although the effect of in vitro PLP sup derline normal value to an elevated or plementation was not examined by pathological level. While both Jung and them, the authors did attempt to explain his colleagues27,28 and Lacour’s group37'38 their results in terms of the depletion of have recommended PLP be added to the plasma pyridoxine during long-term dial reaction mixture if one is to determine ysis. Subsequently, Dobbelstein et al14 correctly the aminotransferases, espe first presented evidence for B6 defi cially AST, in patients with kidney trans ciency in erythrocytes in uremia and plants, and thus to diagnose liver disease Stone and his colleagues demonstrated in such patients, their findings suggest clearly not only the decreased plasma strongly that serum AST and ALT ami AST7° levels but showed decreased notransferase assays on hemodialysis plasma PLP72 levels in uremia. The same patients as well should be supplemented group71 in 1977 found the mean plasma with PLP if liver disease is to be clearance rate of uremic patients to be detected. More recently, Hafkenscheid almost two-fold greater than for control et al19 have demonstrated the type of patients. More recently, Lacour et al37,38 drug treatment, e.g., cyclosporin or have documented further the decreased azathioprine, may effect the percentage of plasma PLP levels in undialyzed and AST and ALT stimulation observed in 90 VANDERLINDE renal allograft patients in contrast to nor ability of such measurements in healthy mal patients. individuals.24 Since B6 deficiency is observed in Miscellaneous alcoholics,43,44 in some significant per centage of hospitalized patients21 and in Several patient cases in which serum apparently healthy people over age 64,20 AST was complexed with immunoglobu these individuals will show PLP stimula lin G (IgG) have been reported36,76 as tion of their serum aminotransferase well as one patient whose serum con enzymes. tained complexes between AST and both Patients with liver disease show lesser IgA and IgG.48 The latter patient suf activation with PLP of AST activity but fered from lung cancer with metastasis to not ALT activity than patients with heart the liver. Nagamine and Okochi48 were disease.43 AST isoenzyme measurements able to demonstrate that only c-AST in the form of a mitochondrial AST/total binds to IgG, whereas IgA binds to both AST ratio may discriminate alcoholic c-AST and m-AST. While serum AST- hepatitis from all other hepatic dis IgG complexes have been demonstrated eases.49,50 Unconfirmed studies suggest in both healthy and diseased individuals, the combination of PLP activation and and have been associated with various AST isoenzyme measurements may aid liver diseases, their clinical significance in the diagnosis of hepatoma.33 In renal is unclear. dialysis patients including transplant Recently, Leklem and Shultz40 have patients, it may be desirable to measure shown for the first time that exercise in the aminotransferases with added PLP in the form of long distance running dra order to reflect better the cytolytic state matically alters plasma levels of PLP and of the liver.28,34,37,38 Table I summarizes total B6. They speculate that the signifi the effect of PLP on aminotransferase cant changes in vitamin B6 metabolites assays in the various liver diseases and found were related to an increased need conditions described. for cofactor for gluconeogenesis. In conclusion, although PLP activa tion per se does not provide clear cut Summary improved diagnostic value of AST and ALT in liver diseases, it is recommended The literature relative to PLP and the that AST and ALT be included in all rou measurement of aminotransferase (AST tine AST and ALT measurements in and ALT) activity has been reviewed in view of its inclusion in the IFCC refer detail recently by Rej.57 In 1979 Camp ence methods for AST5 and ALT6 and bell and Sanderson9 summarized the their adoption into the National Refer published work on the diagnostic signifi ence System for the Clinical Labora cance of PLP supplementation on ami tory.11 notransferase assays, thus this review has focused on findings since their publica References tion. The plasma PLP level in normal indi 1. A a ch , R. D., S z.m u.v ess, W ., M o slev , J. W ., Hol- l in g e r , F. B., K a h n , R. A ., S t e v e n s , C. E., viduals clearly relates inversely to the E d w ard s, V. M . and W e r c h , J.: Serum Alanine degree of in vitro stimulation of serum Aminotransferase of Donors in Relation to the AST and ALT.77 PLP added in vitro Risk of Non-A, Non-B Hepatitis in Recipients. New. E n g . J. Med. 304:989-994, 1981. increases the reference values for AST 2. A lter, H . J., P u r c el l , R. H ., H o lla n d , P. V., but does not decrease the biological vari A ll in g , D. W. and K o z io l , D. E.: Donor Trans PYRIDOXAL PHOSPHATE AND TRANSAMINASES IN LIVER DISEASE 91

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