Biochemical Laboratory Tests in Viral Hepatitis

Transcription

1 Bull. Org. mond. Snte' Bull. Wid Hith Org Biochemicl Lbortory Tests in Virl Heptitis nd other Heptic Diseses Evlution nd Follow-Up * FERNANDO DE RITIS,1 GlUSEPPE GIUSTI,2 FELICE PICCININO8 & LUIGI CACCIATORE 3 The differentil dignosis between virl heptitis nd other liver diseses (prticulrly obstructive jundice) is often difficult on purely clinicl grounds. Dmge to the liver cuses chnges in the pttern of the serum enzymes nd this hs led to the development in recent yers of number of enzyme tests. The uthors hve mssed evidence to show tht the most useful of these is determintion of the levels of serum glutmic oxlcetic nd serum glutmic pyruvic trnsminse (SGOT nd SGPT), coupled with clcultion of the SGOT/SGPT rtio. It is chrcteristic of virl heptitis tht both levels re gretly d, but the SGOT/SGPT rtio, ly greter thn one, flls considerbly below his figure. In few cses of obstructive jundice, the serum trnsminse picture my initilly resemble tht in virl heptitis, but the differentil dignosis cn be estblished by repeting the determintions t intervls. Other enzyme tests, such s determintion of lkline phosphtse nd leucylminopeptidse, my be used to confirm the biliry obstruction. Floccultion tests nd electrophoretic determintion of the plsm protein picture, while of limited vlue in the dignosis of cute virl heptitis, re useful in conjunction with the serum trnsminse test for ssessing the ctivity of the disese nd ny tendency to progress towrds " ctive " chronic heptitis or post-heptic cirrhosis. The dignosis of virl heptitis cnnot esily be confirmed by liver function tests (" loding " tests) or by serum iron determintions. The technicl difficulties mke these methods unsuitble for routine use nd, in ddition, they re not sufficiently specific. Moreover, determintion of serum bilirubin nd of the vrious bilirubin frctions of the blood does not help to clrify the dignosis except in very rre cses. For these resons, ttention hs to be concentrted on two groups of dignostic tests: () those tht exmine the chnges in protein levels frequently ccompnying virl heptitisdetermintion of the plsm protein pttern, floccultion tests, Jirgl test; * Bsed on working pper presented by Professor F. De Ritis to the WHO Expert Committee on Heptitis, Genev, 1-16 December Director, Clinic of Infectious Diseses, University of Nples, Itly. I Chief Assistnt, Clinic of Infectious Diseses, University of Nples, Itly. 3Assistnt, Clinic of Infectious Diseses, University of Nples, Itly. (b) those tht derive from the concept of the so-clled "enzymo-plsmtic syndrome" nd consist in determining the level of certin plsm enzyme ctivities. PLASMA PROTEIN PATTERN AND RELATED TESTS Tests relted to the existence of hypergmmglobulinemi In cute virl heptitis with reversible heptic lesions, moderte fll in plsm lbumins with n eqully moderte in the f- nd y-globulins is noted, wheres in cses of heptitis with cute irreversible lesions (cute trophy of the liver), the fll in the lbumins is ccompnied by prllel fll in the - nd :-globulins nd is ssocited with n in the y-globulins nd with generl hypoproteinemi with hypofibrinogenemi. The picture is quite different in the cse of obstructive jundice in which there is n in the - nd P-globulins but y-globulin level, with n in fibrinogen. These conditions re

2 6A F. DE RrM AND OTHERS found in reltively recent obstructive jundice; when the jundice persists for some time, however, signs of mrked functionl heptic dmge nd of reticulr stimultion lso pper, nd these my be the forerunners of biliry cirrhosis (Viollier, 1957). Consequently, it is only in recent jundice tht the presence of hypergmmglobulinemi rgues in fvour of dignosis of heptitis while gmmglobulinemi is evidence of obstructive jundice. However, determintion of the protein picture, even by the simplest electrophoretic methods, is not routine dignostic test; moreover, it provides only indirect pointers nd is not specific. Consequently, the use of this test is not recommended except for bospitlized ptients in whom there is suspicion of tendency towrds chronicity or post-necrotic cirrhosis. The informtion given by the protein pttern is thus of little prcticl vlue for differentil dignosis between virl heptitis nd obstructive jundice. Tests indirectly reveling chnge in the protein pttern: floccultion tests In the pst, floccultion tests hve plyed lrge prt in the dignosis of nicteric heptitis nd in the identifiction of clinicl pictures tending towrds " ctive " chronic heptitis or post-necrotic cirrhosis. Nevertheless, they hve certin importnt limittions, such s the subjectivity, nd therefore inccurcy, of the opticl determintion of serum turbidity nd the bsence of dequte stndrds (resulting, for exmple, in considerble vritions in the results of comprtive thymol-turbidity tests crried out in different lbortories). The need to dopt certin technicl improvements so s to obtin mximum sensitivity, specificity nd reproducibility, leds to the trnsformtion of simple tests into complicted techniques with ll the disdvntges this entils. In ddition, it is generlly necessry to tke into ccount the limittions resulting from the lck of specificity. The most chrcteristic exmple is tht of long-stnding obstructive jundice in which the progression of the heptic lesions renders the floccultion tests positive exctly s in cute or subcute virl heptitis. Furthermore, in firly lrge number of cses of icteric or nicteric virl heptitis, the floccultion tests re negtive. Recourse to floccultion tests lone, therefore, hs now been superseded nd is no longer to be recommended. When floccultion tests re combined with enzyme tests, they do not dd very much to the dignostic vlue of the ltter in cute cses, but re useful s indictors of the " ctivity " nd progressive nture of pprently cured heptitis cses. The Jirgl test This is floccultion test for determining plsm frctions tht re soluble in sulfoslicylic cid but insoluble in phosphotungstic cid. A positive result is useful pointer to dignosis of obstructive jundice. The test gives 25 % flse positive results in virl heptitis nd 1% flse negtive results in obstructive jundice. It is of limited vlue in differentil dignosis (Jirgl, 1957; Coltorti et l., 1963). ENZYME TESTS A new line of ttck in dignosis, the usefulness of which hs been confirmed during the pst nine yers, is bsed on the utiliztion of physiopthologicl phenomenon chrcteristic of virl heptitis, nmely, the ppernce in the serum of enzyme ctivities tht re prcticlly, bsent in subjects, or n -often very considerble-in enzyme ctivities whose rte is extremely low. This hs been termed the "enzymo-plsmtic syndrome" (De Ritis et l., 1955, b). The number of enzymes present, or pthologiclly d, in the plsm during virl heptitis, nd their comprtive behviour in two other types of liver disese (obstructive jundice nd heptic cirrhosis) re given in Tble 1. The first question to be considered is whether the enzymes studied re etiologiclly specific. On this point the reply is negtive. The nture of the enzymes relesed into the blood plsm from heptic cells whose physiologicl structure hs undergone pthologicl chnge (rnging from simple modifiction of cellulr permebility to necrosis) is relted not to the etiologicl gent but to the type of lesion. In heptitis, the principl lesion is necrosis nd similr relese of enzymes occurs in other non-virl types of necrosis (ischemic, toxic). The second problem is tht of the orgnospecificity of the enzymes. The idel would be to be ble to detect in the plsm the ppernce of enzyme ctivities originting exclusively in the liver. There re some enzymes tht fulfil this condition but their detection-for resons tht will be referred to lter-does not represent ny gret dignostic dvnce. On the other hnd, there re certin

3 BIOCHEMICAL TESTS IN VIRAL HEPATITIS 61 TABLE I ENZYMES PRESENT OR PATHOLOGICALLY INCREASED IN THE PLASMA IN VIRAL HEPATITIS, OBSTRUCTIVE JAUNDICE AND HEPATIC CIRRHOSIS Enzyme Chnge in plsm level in: Virl heptitis Obtrciv_ Obstructiveiudie epti_irhoi jundice Heptic cirrhosis Trnsminses sprtic-ketoglutric trnsminse (SGOT) lnine-ketoglutric trnsminse (SGPT) Glycolysis enzymes Other gluclde metbolism enzymes Citric cycle enzymes Ure cycle enzymes Peptidses Esterses phosphoglycomutse phosphohexose isomerse phosphofructose kinse fructose-1 6- diphosphte ldolse glycerldehyde-3- phosphte dehydrogense triose phosphte isomerse enolse pyruvic kinse lctic dehydrogense fructose kinse fructose-i-phosphte ldolse sorbitol dehydrogense -glycerophosphte dehydrogense glucose-6-phosphte dehydrogense 6-phosphogluconic dehydrogense 5-phosphoribose isomerse trnsketolse isocitric-dehydrogense mlic dehydrogense fumrse rginse ornithine-crbmyl trnsferse leucyl minopeptidse glycyl tyrosinse dehydropeptidse minotripeptidse lkline phosphtse 5-nucleotidse denosine triphosphtse cholinesterse cholesterol esterse desoxyribonuclese Other enzymes A-glycuronidse glucose-6-phosphtse glutthione reductse glutmic dehydrogense considerble considerble considerble considerble considerble considerble moderte lrge Increse moderte nd irregulr slight reduction moderte Increse moderte lrge lrge moderte slight nd Irregulr mrked Increse mrked mrked nd Irregulr moderte lrge moderte nd irregulr red uction moderte slight nd irregulr slight nd irregulr slight nd irregulr reduction slight reduction lrge moderte moderte irregulr moderte moderte slight nd Irregulr Increse slight nd Irregulr slight nd irregulr slight nd irregulr slight Increse slight nd irregulr lrge lrge lrge lrge slight reduction moderte slight nd irregulr slight nd irregulr slight nd irregulr slight reduction slight Increse slight nd irregulr norm moderte Increse moderte slight nd irregulr slight nd irregulr moderte nd irregulr slight reduction slight reduction slight nd irregulr

4 62 F. DE RITIS AND OTHERS enzymes tht hve no bsolute orgno-specificity but whose behviour in virl heptitis is nevertheless chrcteristic. Determintion of serum trnsminses This test ws introduced by De Ritis, Coltorti & Giusti (1955, b) in 1955 nd the sme uthors hve since provided, further, exhustive evidence of its vlue for the dignosis of virl heptitis (De Ritis, Coltorti & Giusti, 1956,b; 1957; 1961; De Ritis, Ascione et l., 1959). Now-fter eight yers of use nd extensive studies ll over the world-the following conclusions my be reched: (1) In cute virus heptitis there is considerble in the ctivities of serum glutmic oxlcetic nd serum glutmic pyruvic trnsminse (SGOT nd SGPT), n which is sometimes of the order of 2-4 % nd which is seldom less thn 1%. The chrcteristic behviour of the trnsminses in heptitis is s follows: considerble in both SGOT nd SGPT ctivities (up to 4 times the figures) with lrge reltive in SGPT with respect to SGOT nd consequent decrese in the SGOT/SGPT rtio to considerbly less thn one. Normlly this rtio is greter thn one. (2) The sme behviour is lso seen during nicteric heptitis (this is why, when the ltter is suspected, prticulrly in communities, it is esy to obtin confirmtion by determining the trnsminse vlues). (3) The in the trnsminses precedes the ppernce of jundice nd possible chnge to positive result in the floccultion tests. In this connexion it my be mentioned tht liver biopsies in subjects with high trnsminse level of the heptic type but without evident symptoms or jundice hve reveled the presence of typicl ctive heptitis lesions. It is not until 2 to 3 dys lter tht jundice my pper in such subjects (Tolentino et l., 1957; Delkeskmp et l., 1959). This finding completely confirms previous ides in regrd to incubtion, for it shows: () tht the disese ctully exists, if only in subclinicl form, three to five weeks before ny clinicl mnifesttion, so tht it is hrdly correct to spek of incubtion during this period; (b) tht in certin cses the length of the period when heptitis is cliniclly evident my be less thn tht of the symptomtic period. (4) The plsm trnsminse vlues re independent of the degree of bilirubinemi, the serum protein levels, nd the behviour of the floccultion tests, since ll these tests re n expression of vrious pthogeniclly independent phenomen. (5) During virl heptitis, the plsm trnsminse levels my show vritions which reflect the subsequent course of disese. (6) During convlescence while the bsolute trnsminse vlues re returning to, the SGOT/SGPT rtio my remin low for one or more weeks which sometimes mkes possible retrospective dignosis of virl heptitis. (7) The mere finding of n SGOT/SGPT rtio below one cnnot be considered s chrcteristic of virl heptitis if the bsolute figures for the two enzyme ctivities re or only modertely d. In no other liver disese (heptitis with bcteril hepto-cholngitis, cirrhosis of the liver, neoplsm) or even in diseses not ffecting the liver hve plsm trnsminse ptterns been observed similr to those seen in virl heptitis, prt from few rre exceptions (in extensive neoplsm of the liver with severe necrosis nd in moebic heptitis during the erly colliqution stge). In some cses of very recent obstructive jundice (the cuse of the biliry obstruction is immteril), the sudden biliry stsis is ccompnied by retrogrde ction of the bile on the liver cells cusing dmge to the ltter which my led to necrosis. As consequence, plsm trnsminse levels similr to those of virl heptitis my be found, lthough the bsolute figures re generlly much lower. In such specil cses, obstructive jundice my esily be differentited from virl jundice by the repetition t close intervls of plsm trnsminse tests, even in the bsence of clinicl criteri or other lbortory dt, such s high lklinephosphtse blood level or hypercholesterolemi. In obstructive jundice, the initilly high trnsminse figures tend to decrese nd, in prticulr, the SGOT/SGPT rtio becomes ; this does not occur in virl jundice, where the chrcteristic very low rtio (bout.5-.6) persists while the bsolute concentrtions of the two enzymes remin elevted nd sometimes further. Appliction of these criteri hs mde it possible, even in subjects known to be suffering from cholelithisis to demonstrte tht the jundice is due to

5 BIOCHEMICAL TESTS IN VIRAL HEPATIS 63 heptitis with initil symptoms chrcterized by pin in the right hypochondrium nd not to the presence of the clculi. The bove-mentioned criteri, bsed on our personl experience of more thn eight yers, hve been confirmed by mny other workers. Further confirmtion hs come from the evlution of serum trnsminse ptterns in recent series of 263 icteric ptients. The 263 ptients were composed of 195 unselected cses of virl heptitis, 23 cses of bcteril cholngiolitis, nd 45 cses of cholesttic jundice. The collected dt re summrized in Fig. 1-6 nd re clerly in ccordnce with the previous conclusions. To mke cler the usefulness in the dignosis of virl heptitis of the simultneous determintions of SGOT, SGPT nd the SGOT/SGPT rtio, we constructed the histogrm shown in Fig. 7. The serum trnsminse vlues found in the 195 cses of virl heptitis were initilly divided into two min groups. In the first group, we collected ll the cses tht showed very lrge bsolute in serum trnsminse (prticulrly those with SGPT vlues >71umol/ml of pyruvte formed); in the second group, we collected those cses in which the in the serum trnsminses, lthough considerble, ws lower thn tht in the first group. Ech of these two groups ws then divided into two subgroups, ccording to the vlue of the SGOT/SGPT rtio (between.99 nd.7, or less thn.7). The men vlues in ech of the four groups re shown below the bsciss in Fig. 7. The men vlues of the SGOT/SGPT rtio re very much less thn one in ech group. This low vlue of the rtio mkes the dignosis very esy in the first nd second groups of cses (comprising 77 % of the totl) becuse it is lwys ssocited with very high serum trnsminse levels. In the third group (2% of the totl) the in the trnsminses is less pronounced, but the dignosis is lso esy becuse of the very low vlues of the rtio (men=.49). Only in 2.1 % of the cses is the dignostic vlue of the test impired becuse reltively low vlues of the SGOT/SGPT rtio (verge=.82) re ssocited with rther smll s in the serum trnsminses. The usefulness of simultneous determintion of the SGOT nd SGPT levels nd the SGOT/SGPT rtio in the differentition of cholesttic jundice from virl heptitis is evident from Fig. 8, which shows the men vlues found in 45 cses of cholesttic jundice, divided into four groups. In the first group-which represents 77.8 % of the totl-re included cses with bsolute figures of serum trnsminses, or with vlues only slightly d, nd with n SGOT/SGPT rtio greter thn one. In the second group-1 5.6% of the totl-re included cses with the trnsminses slightly d nd the rtio lower thn one. The third group comprises the cses in which moderte in trnsminse levels is ssocited with n SGOT/SGPT rtio greter thn one. In these first three groups, therefore, the differentition from virl heptitis is unmistkble. It is only in the fourth group-4.4 % of the totl-where gretly d serum trnsminse levels re ssocited with rtio decidedly lower thn one tht no useful dignostic conclusions cn be drwn. Our observtions on cses of bcteril cholngiolitis show in ll cses low trnsminse figures ssocited with n SGOT/SGPT rtio ner to one. It is therefore esy to mke differentil dignosis between this disese nd virl heptitis, but it is not possible to differentite it from cholesttic jundice. On combining the informtion from Fig. 5 nd 6, it is pprent tht simultneous determintion of the SGOT nd SGPT levels nd of the SGOT/SGPT rtio mkes the dignosis esy in more thn 97 % of the cses of virl heptitis nd in more thn 95 % of the cses of cholesttic jundice. The differentil dignosis in the smll percentge of the cses of cholesttic jundice in which the behviour of the serum trnsminse levels is not conclusive cn be estblished by mens of further repetitions of the SGOT nd SGPT tests. In fct, trnsminse figures tht re initilly high show quick decrese, while bilirubin vlues remin high or further (Fig. 9). Other plsm enzyme tests studied for dignosing heptocellulr lesions of the necrotic type Among the very numerous enzymes tht re found in the plsm during virl heptitis (see Tble 1), certin ones re prticulrly suitble for dignosing heptocellulr lesions of the necrotic type. These re: fructose-1,6-diphosphte ldolse, phosphohexose isomerse, phosphoglycomutse, triose-phosphte isomerse, isocritric dehydrogense (Bruns & Puls, 1954; De Ritis, Giusti & Coltorti, 1957,b; Giusti, 1962; Wolfson et l., 1958).

6 i 64 FIG. 1 SGOT AND SGPT LEVELS AND SGOT/SGPT RATIOS IN NORMAL SUBJECTS AND IN PATIENTS WITH DIFFERENT HEPATIC DISEASES WITH JAUNDICE v r._ 8 H km E 2 14 F. DE RIM AND OTHERS IU, - r FIG. 2 FREQUENCY DISTRIBUTION OF SGOT LEVELS IN 195 CASES OF ACUTE VIRAL HEPATITIS o k 2. E 1 5 z I, 1 _ 7 j, 5, we _ o,.i : ). 3 :o : -6. -s oo CM coi WHO 5168._ I.. I I I I wmo 5169 p mol of ketoccid formed by 1 ml of serum in 15 min t 37 'C 16 _ SGOT SGPT SGOT/SGPT rtio (men vlues In ech group) FIG. 3 FREQUENCY DISTRIBUTION OF SGPT LEVELS IN 195 CASES OF ACUTE VIRAL HEPATITIS 3t, 25 -.E z I I 5 A V l p mol of ketoocid formed by 1 ml of serum in 15 min t 37 C WH 51 7

7 BIOCHEMICAL TESTS IN VIRAL HEPATITIS 65 FIG. 4 FREQUENCY DISTRIBUTION OF SGOT/SGPT RATIOS IN 195 CASES OF ACUTE VIRAL HEPATITIS FIG. 5 PAIRED VALUES OF SIMULTANEOUSLY DETERMINED SGOT AND SGPT LEVELS IN 195 CASES OF ACUTE VIRAL HEPATITIS A-S. 4 35H 3 - U).@ 25 -o = 2 15k 1 5 I T 1 I T ET 11I. " f l ~~~~~~~~~~~~~~~~~~~~~~~~~~I A SGOT/SGPT rtio WHO 5171 o me.e,c- C 8= SU) - o U "S I- CD ii) S."O mr= r= S I 9. l.1 :2 I , The behviour of ll these enzymes is similr: they show pronounced nd erly in virl heptitis (evenof the nicteric type) but only slight or negligible in the course of jundice cused by cholestsis nd other liver complints. The determintion of these s mkes it possible in lrge number of cses to estblish the differentil dignosis of liver diseses with high degree of probbility. Persistence of high plsm enzymes levels when the bilirubin level hs returned to nd the floccultion test hs become negtive indicte persistent " ctivity " of the heptic disese. Ech line _-. joins pir of vlues determined in single ptient.

8 66 F. DE RMTIS AND OTHERS FIG. 6 PAIRED VALUES OF SIMULTANEOUSLY DETERMINED SGOT AND SGPT LEVELS IN 45 CASES OF OBSTRUCTIVE JAUNDICE FIG. 7 SGOT AND SGPT LEVELS AND SGOT/SGPT RATIOS IN 195 CASES OF ACUTE VIRAL HEPATITIS 12 '8 ( 8 U 1.S.C-, c p- C IE c *_C. 8 E u z S g- o, ) -' I 12 2 S V l \o. IC' E.: %6-~ 12 cses 5 cses 39 cses 4 cses 52.3% 25.6% 2.% 2.1% -Ico =1, n 3. %.5 I-.o 3C CD - ccet uerv i jy ~ l s CD ~ SGOT SGPT SGOTISGPT rtio (men vlues in ech group) Ech line _ joins pir of vlues determined in single ptient. Hepto-specific enzyme tests: fructose-l-phosphte ldolse; ornithine-crbmyl trnsfirse; sorbitol dehydrogense Certin other enzymes tht re not present in ny orgn except the liver re found in remrkbly d quntities in the serum during virl heptitis, but much less in obstructive jundice (fructose-l-phosphte ldolse, ornithinecrbmyl-trnsferse, sorbitol-dehydrogense) while they do not t ll in extr-heptic diseses (Wolf et l., 1957; Giusti et l., 1961; Reichrd, 1957). The hepto-specificity of these enzymes, i.e., their property of being confined exclusively to the liver, is dignostic dvntge only in theory. In prctice, the determintion of these enzymes does not offer ny rel dignostic dvntge, since the in the trnsminses nd in fructose-1,6-diphosphte ldolse during myocrdil infrction nd in ptients suffering from musculr distrophy does not

9 BIOCHEMICAL TESTS IN VIRAL HEPATITIS FIG. 8 SGOT AND SGPT LEVELS AND SGOT/SGPT RATIOS IN 45 CASES OF OBSTRUCTIVE JAUNDICE u 12 es 1 I.S *1 cs 8 E 2! 6. I14._ 4 _ EC2 G FIG. 9 SGOT, SGPT AND BILIRUBIN LEVELS IN A CASE OF OBSTRUCTIVE JAUNDICE WITH INITIAL TRANSAMINASE VALUES SIMULATING THE VIRAL HEPATITIS PATTERN 12 E 1 g 8, 6 SE 2 _ 25 wen 51?h 77.8 % 15.6% 2.2% 4.4%... SGOT -*- SGPT -bbilirubin U O SGOT SGPT - - ~~~~~~~. _- SGOT/SGPT rtio (men vlues in ech group) present problem in the study of the liver diseses. Determintion of the concentrtion of the orgnospecific enzymes is of use only in specil cses, e.g., in the dignosis nd follow-up of virl heptitis (whether icteric or not) ssocited with musculr dystrophy (where high trnsminse or fructose-1,6- diphosphte ldolse levels might be of musculr origin nd would not justify dignosis of virl heptitis). Aprt from such extremely rre borderline cses, determintion of the hepto-specific enzymes is not dvisble in the study of virl heptitis becuse of their lower sensitivity nd their erly decrese in the plsm even while the disese is still in the ctive phse (jundice). VI Tests on plsm enzymes used for the dignosis of jundice of the cholesttic type A certin number of tests on plsm enzymes re vluble both for the dignosis of obstructive jundice (becuse of the considerble of these enzymes in the plsm) nd lso for the dignosis of virl heptis. A comprison between the humorl ptterns found in virl heptitis nd in cholesttic jundice is shown in Tble 2. While these tests re not of ny rel vlue in cses where the results of the trnsminse test re chrcteristic, they my be of considerble ssistnce in the smll number of cses where the results of the lbortory tests re not sufficiently typicl (e.g., where there is only in the 67 levels of the two trnsminses, where the floccultion tests re negtive, or where there is no pprecible chnge in the protein pttern). It is difficult to differentite these cses from obstructive jundice, prticulrly if there is high bilirubinemi nd mrked decolortion of the stools (hypocholi). Under such conditions, which re, however, extremely rre, the discovery of distinct in enzyme ctivity is indictive of obstructive jundice; on the other hnd, the finding of figures, or t ny rte of figures not significntly

10 68 F. DE RITIS AND OTHERS TABLE 2 COMPARISON BETWEEN THE HUMORAL PATTERNS CHARACTERISTIC OF VIRAL HEPATITIS AND OF CHOLESTATIC JAUNDICE Humorl pttern chrcteristic of: Virl heptitis Cholesttic Trnsminses mrked with low SGOT/ with high SGOT/ SGPT rtio SGPT rtio Alkline phosphtse mrked nd leucyl minopeptidse Serum Iron mrked Serum cholesterol or slight mrked reduction Floccultion tests positive negtive Serum globulins Increse In long-stnding cholesttic jundice, n of serum globulins with positive floccultion tests cn be observed. different from, my indicte dignosis of virl heptitis. The following enzymes belong to this group: () Alkline phosphtse. In cholesttic jundice, serum lkline phosphtse shows mrked, chiefly in complete nd long-stnding obstruction (Bodnsky, 1933; Roberts, 193; Coltorti et l., 1963); the s re much less in virl heptitis nd do not occur in nicteric heptitis. The following figures show the dt found in this condition in our observtions: Norml subjects Virl heptitis Cholesttic jundice 1.1 i.54 units 3.25 ± 1.22 units 16.3 ± 9.35 units Determintions were crried out by the method of Bessey, Lowry & Brock (1946) nd the results were reported s Bessey-Lowry units (1 unit= 1,tmol/litre/hour of split substrte). (b) 5-nucleotidse. The behviour of this enzyme is similr to tht of lkline phosphtse but it is more specific, since no is seen in skeletl diseses (Dixon & Purdom, 1954; Young, 1958). (c) Leucylminopeptidse. The ctivity of this enzyme lso s considerbly in cholesttic jundice, prticulrly if cholestsis is complete nd hs existed for long time. On the other hnd, the is much less in virl heptitis (Goldbrg & Rutenburg, 1958; Bnks et l., 196; Giusti & Piccinino, 1963). Determintion of the vrious bilirubin frctions of the blood is of prcticlly no dignostic ssistnce. The pttern given for the forms of virl heptitis, whether icteric or nicteric, holds lso for the forms with cholngiolitis or cholestsis which sometimes cn hrdly be differentited cliniclly from cholesttic jundice (choli). It would seem, ccording to the present literture -nd this is lso our conviction fter experience extending over nerly 1 yers-tht it is essentil in studying liver disese to determine the ctivities of both trnsminses (SGOT nd SGPT, s well s the SGOT/SGPT rtio), for if only one were mesured this would give much more limited dignostic informtion (prticulrly in the cse of the SGOT) nd might thus become source of error. EPIDEMIOLOGICAL AND CLINICAL APPLICATIONS OF DETERMINATIONS OF PLASMA TRANSAMINASE LEVELS Identifiction of cses of nicteric virl heptitis, or of the pre-icteric stges of virl heptitis The determintion of the serum trnsminse levels in subjects living in closed environments in which cses of icteric virl heptitis hve been reported, or in subjects otherwise exposed to the infection, llows the identifiction of nicteric cses of the disese s well s of the pre-icteric stges. By conducting n investigtion long these lines, we were ble to demonstrte tht in some environments the cses of nicteric virl heptitis gretly outnumber the icteric cses, the reltive frequency rnging from 3:1 to 3:1; this is especilly so in communities of children (De Ritis, Mllucci et l., 1959; Giusti et l., 1959; Coltorti et l., 1961). The identifiction of the cses of nicteric heptitis in the community lso permits isoltion of virus crriers. These re the subjects most likely to be responsible for the spred of the disese, prticulrly s they re often subcliniclly ill. Identifiction of suspected crriers of virl heptitis mong blood donors When determintion of trnsminse levels in prospective blood donors revels modertely d figures with persistent lowering of the SGOT/SGPT rtio, this clls ttention to possible

11 BIOCHEMICAL TESTS IN VIRAL HEPATITIS 69 heptitis. Such subjects cn then be rejected s blood donors. A trnsminse response of this type does not by itself show whether the subject hs hd virl heptitis in the pst or whether the disese is incipient. The problem cn esily be resolved by repeted determintions which, in the ltter cse, will revel incresing vlues for SGOT nd SGPT with the SGOT/SGPT rtio flling well below 1. SGOT nd SGPT determintions should be compulsory for ll blood donors. There re not sufficient dt regrding the behviour of the trnsminses in unrecognized virl heptitis crriers. Evlution of the drug tretment of virl heptitis SGOT nd SGPT determintions repeted every four or five dys in cliniclly selected subjects who hve been suffering from recent jundice nd who re found initilly to hve similr serum trnsminse nd bilirubin levels my supply vluble informtion on the effectiveness of drugs in the tretment of virl heptitis. The gret clinicl vribility of the disese mkes it impossible to rech decision solely on clinicl bsis. Fig show the results of controlled experiment on the effectiveness of prednisone (l-cortisone) in virl heptitis (De Ritis et l., 1964). The results of this study re not sttisticlly significnt lthough the men vlues of the serum trnsminse nd bilirubin levels re somewht lower for the prednisone group thn for the plcebo group. The lck of sttisticl significnce is due to the fct tht the prednisone group includes remrkble number of cses tht seem to be completely unffected by the therpy. On the other hnd, the plcebo group includes certin number of cses tht show n erly, spontneous improvement indistinguishble from tht observed in the cses pprently responding to prednisone tretment. Trnsminse determintions in the ssessment of the ctivity of pprently cured virl heptitis In most cses, the pthologicl trnsminse figures become 3-6 weeks fter the ppernce of jundice, wheres the SGOT/SGPT rtio my remin considerbly lower thn 1 for period vrying from one to severl weeks nd my persist for some time fter the individul levels hve returned to. The discovery of trnsminse levels in successive tests in subject who hs suffered from icteric heptitis but no longer shows jundice my be tken s evidence of cure, even if the SGOT/ SGPT rtio remins less thn 1, provided tht the clinicl findings lso point in the sme direction (return of the liver size to, disppernce of splenomegly, iztion of the protein picture, etc.). In other cses, the fll in the trnsminse levels does not proceed smoothly but is interrupted by fresh rises, probbly corresponding to new outbreks of heptic necrosis; these fresh rises in the figures for the two enzyme ctivities re frequently ssocited in our experience with decrese in the SGOT/SGPT rtio, wheres fll in the bsolute.figures is ssocited with n in this rtio. In limited number of cses, while the jundice disppers nd the generl condition improvesprtly becuse of better intestinl bsorption due to the re-estblishment of bile excretion into the intestine-the trnsminse vlues remin pthologiclly high nd their rtio below 1. This my occur either in the presence of dysproteinemi with positive floccultion tests or in bsence of other FIG. 1 PERCENTAGE DECREASE IN SGOT LEVELS IN VIRAL HEPATITIS PATIENTS TREATED WITH PREDNISONE AND WITH A PLACEBO - men vlues in 25 cses treted with plcebo -- men vlues in 37 cses treted with prednisone SGOT: p mol of ketocid formed by I ml of serum in 15 min t 37 C (men of bsolute vlues)

12 7 F. DE RIllS AND OTHERS FIG. 11 PERCENTAGE DECREASE IN SGPT LEVELS IN VIRAL HEPATITIS PATIENTS TREATED WITH PREDNISONE AND WITH A PLACEBO FIG. 12 PERCENTAGE DECREASE IN SERUM BILIRUBIN IN VIRAL HEPATITIS PATIENTS TREATED WITH PREDNISONE AND WITH A PLACEBO - men vlues In 31 cses treted with plcebo men vlues in 22 cses treted with plcebo -- men vlues in 34 cses treted with prednisone -- men vlues in 23 cses treted with prednisone Q SGPT: t& mol of ketocid formed by I ml of serum in Q serum bilirubin in mg/1 ml (men of bsolute vlues) 15 min t 37 C (men of bsolute vlues) humorl signs of ctive disese; these b figures my then be the prelude to new ttcks of jundice (relpsing heptitis). In other cses, pthologicl figures my persist, the vlues fluctuting over firly long periods without recurrence of jundice, while the liver s once more in size. Liver biopsies crried out on subjects with these b enzyme levels hve reveled distinct signs of ctive nd progressing heptitis. For this reson, one should reserve judgement of the prognosis, since it is in such cses tht heptitis most frequently becomes chronic or tht post-necrotic cirrhosis tends to develop. In our experience, the trnsminse test reflects better thn ny other determintion the progress nd ctivity of heptitis in cse tht is pprently cured nd is cliniclly silent. CONCLUSIONS It is often very difficult to differentite virl heptitis cliniclly from obstructive jundice. Extensive studies of the mny enzyme tests proposed in vrious prts of the world during the lst 8 yers hve led to the conclusion tht the " two-trnsminse test" proposed by De Ritis, Coltorti & Giusti in 1955 is still the best, since these serum enzymes (SGOT nd SGPT) show pttern tht is highly chrcteristic of virl heptitis nd llows dignosis in s mny s 98 % of cses. In cute virl heptitis, there is considerble in the ctivity of the two trnsminsesn sometimes of the order of 2-4 %Y nd not usully less thn 1 %. The chrcteristic behviour of the trnsminses in heptitis is s follows: considerble in both SGOT nd SGPT ctivities (up to 4 times the figures) with lrge reltive in SGPT s compred with SGOT nd consequent decrese in the SGOT/ SGPT rtio to figures considerbly less thn one. The in the trnsminse levels precedes the ppernce of jundice nd possible chnge in the floccultion tests to positive. In this connexion, it hs been shown by liver biopsies in subjects with high trnsminse level of the heptic type but without evident symptoms of jundice tht typicl

13 BIOCHEMICAL TESTS IN VIRAL HEPATITIS 71 heptitis lesions re present nd fully ctive. It is not until 2 to 3 dys lter tht jundice ppers in such subjects. SGOT/SGPT rtios just below 1 cnnot be considered s chrcteristic of virl heptitis if the bsolute figures for the two enzyme ctivities re or only modertely d. In no other liver disese (prt from few rre exceptions), or even in diseses not ffecting the liver, re plsm trnsminse levels observed similr to those seen in virl heptitis. In some cses of very recent obstructive jundice, sudden biliry stsis is ccompnied by retrogrde ction of the bile on the liver cells leding to necrosis nd trnsminse picture of the virl heptitis type. In such cses, repetition of the plsm enzyme tests t short intervls will fcilitte the differentil dignosis even in the bsence of clinicl criteri or other lbortory dt. In few doubtful cses, the differentil dignosis between heptic jundice nd obstructive jundice clls for dditionl investigtions, e.g., determintion of the levels of lkline phosphtse nd leucylminopeptidse; floccultion tests; or determintion of the protein picture, the serum iron level, or the sedimenttion rte. The trnsminse test is vluble not only in the clinicl study of virl heptitis-dignosis nd followup of cses, evlution of the " ctivity " of subcute nd chronic heptitis, effectiveness of drug tretment -but lso s n epidemiologicl id in detecting nicteric cses of heptitis by mss screening of popultion groups nd in identifying possible virus crriers mong blood donors. The persistence of n b elevtion of the two trnsminse levels, or fluctution of these levels, combined with lowered SGOT/SGPT rtio is sensitive index of the " ctivity " of the disese. Where there re considerble vritions in the ctivity of the disese, ssocition of the trnsminse test with floccultion tests nd plsm protein determintions is useful procedure. POSTSCRIPT The WHO Expert Committee on Heptitis (1964) endorsed the use of the unit recommended by the Commission on Enzymes of the Interntionl Union of Biochemistry (1961) nd pproved by the Clinicl Chemistry Commission of the Interntionl Union of Pure nd Applied Chemistry. (IUB unit) is described s follows: This unit " One unit (U) of ny enzyme should be defined s tht mount which will ctlyse the trnsformtion of 1 micromole of substrte per minute, or, where more thn one bond of ech substrte molecule is ttcked, 1 micro-equivlent of the group concerned per minute, under defined conditions. Where two identicl molecules rect together, the unit will be the mount which ctlyses the trnsformtion of 2 micromoles per minute. The temperture should be stted, nd where prcticble should be 25 C. The other conditions, including ph nd substrte concentrtion, should be optiml. In order to void inconvenient numbers, terms such s milli-unit (mu), kilo-unit (ku), etc., my be used." Serum enzyme concentrtions re usully stted in terms of U/1 or mu/ml. For comprision with our previous dt, our results hve been given in terms of jumol/ml of trnsformed substrte per 15 min t 37 C. These vlues my esily be converted into IUB units by mens of the following formul: n x 1 N= 15 where N = the number of U/1 or mu/ml nd n = the number of,umol/ml formed in 15 minutes t 37C. RESUMIt Le dignostic differentiel entre l'heptite virle et d'utres ffections du foie - l'ictere pr retention en prticulier - est souvent difficile d'pres les seuls criteres cliniques. Les l6sions du foie entrinent des chngements des enzymes s6riques, ce qui conduit A l mise u point de tests enzymtiques. Les uteurs, pres 1 ns d'exp& rience, sont rriv6s A l conclusion que les tests les plus pr6cis sont ceux qui portent sur les trnsminses, oxlcetique et pyruvique (TGO et TGP) vec clcul du rpport de l'une sur l'utre. L'el6vtion du niveu des trnsminses, qui peut tteindre 4 fois le niveu, crcterise l'h6ptite virle, tndis que le rpport, ement de 1, s'bisse notblement. Ces tests ont permis un dignostic exct dns 98% des cs. I1 rrive que, dns des cs d'ictere pr r6tention, le tux des trnsminses se rpproche, u d6but, de celui que l'on observe dns l'h6ptite virle, mis les differences

14 72 F. DE RIT AND OTHERS s'ccentuent si l'on repete les tests intervlles de temps. D'utres tests enzymtiques, tels que l determintion de l phosphtse lcline et celle de l leucylminopeptidse peuvent etre utiles pour confirmer l'obstruction des voies biliires. Les tests de flocultion, l determintion pr 6lectrophorese des prot6ines plsmtiques, bien que d'importnce mineure pour le dignostic de l'h6ptite virle igue, sont utiles lorsqu'ils sont ssoci6s ux tests des trnsminses seriques, pour suivre l'evolution de l mldie et son cheminement vers l'heptite chronique 6volutive ou l cirrhose post-heptitique. Les tests des trnsminses ont ussi une vleur 6pid6- miologique, en permettnt de depister les cs d'h6ptite nicterique pr exmen de groupes de popultion et d'6ventuels porteurs de virus de l'h6ptite prmi les donneurs de sng. REFERENCES Bnks, B. M., Pined, E. P., Goldbrg, J. A., Rutenburg, A. M. (196) New Engl. J. Med., 263, 1277 Bessey, A. O., Lowry,. H. & Brock, M. J. (1946) J. bio. Chem., 164, 321 Bodnsky,. (1933) J. bio. Chem., 11, 93 Bruns, F. & Puls, W. (1954) Klin. Wschr., 32, 656 Coltorti, M., Di Simone, A., Brbieri, A. M., del Vecchio Blnco, C. (1963) Policlinico, Sez. med., 7, 1596 Coltorti, M., Giusti, G. & Cirillo, C. (1961) Therpeutikon (Pis), 1, 79 Delkeskmp, A., Schmidt, E. & Schmidt, F. W. (1959) Dtsch. med. Wschr., 84, 188 De Ritis, F., Ascione, A., Coltorti, M., Giusti, G. & Mllucci, L. (1959) Eptite virle, Relzione l If Congresso Internzionle di Ptologi Infettiv dell Societ Internzionle per lo studio delle Mlttie Infettive e Prssitrie. Milno 6-1 Mggio 1959: G. Ml. infett., 11, Suppl. De Ritis, F., Coltorti, M. & Giusti, G. (1955) Minerv Med., 1, 127 De Ritis, F., Coltorti, M. & Giusti, G. (1955b) Boll. Soc. itl. Biol. sper., 31, 394 De Ritis, F., Coltorti, M. & Giusti, G. (1956) Minerv Med., 1, 167 De Ritis, F., Coltorti, M. & Giusti, G. (1956b) Recenti Progr. Med., 2, 533 De Ritis, F., Coltorti, M. & Giusti, G. (1957) Clin. chim. Act, 2, 7 De Ritis, F., Coltorti, M. & Giusti, G. (1961) In: Proceedings of the First Europen Symposium on Medicl Enzymology, Miln, 196, Bsel, Krger, pp De Ritis, F., Giusti, G. & Coltorti, M. (1957) Minerv Med., 1, 1958 De Ritis, F., Giusti, G. & Coltorti, M. (1957b) Experienti, 13, 81 De Ritis, F., Giusti, G., Mllucci, L. & Pizz, M. (1964) In: The Third Interntionl Congress of Infectious Pthology, Communictions, Buchrest, 8-11 October 1962, Buchrest, Editur Acdemiei Republicii Populre Romine, pp De Ritis, F., Mllucci, L., Coltorti, M. & Clder, M. (1959) Bull. Wld Hlth Org., 2, 589 Dixon, F. & Purdom, M. (1954) J. clin. Pth., 7, 351 Giusti, G. (1962) Boll. Soc. itl. Biol. sper., 38, 1 Giusti, G., Coltorti, M. & Cirillo, C. (1959) Ml. infett., 11, 16 Giusti, G., Di Simone, A. & Coltorti, M. (1961) Enzymologi, 23, 163 Giusti, G. & Piccinino, F. (1963) Act hepto-splenol. (Stuttg.), 1, 166 Goldbrg, J. A. & Rutenburg, A. M. (1958) Cncer, 11, 283 Interntionl Union of Biochemistry (1961) Report of the Commission on Enzymes, London & New York, Pergmon Press, p. 45 Jirgl, V. (1957) Klin. Wschr., 35, 938 Reichrd, H. (1957) Nord. Med., 58, 116 Roberts, W. M. (193) Brit. J. exp. Pt., 11, 9 Tolentino, P. & Rossi, M. (1957) G. Ml. infett., 9, 552 Viollier, G. (1957) In: Hrtmnn, F. W., Lo Grippo, G. A., Mteer, J. G. & Brron, J., Heptitis frontiers, Boston, Little, Brown & Co., pp WHO Expert Committee on Heptitis (1964) Wld Hlth Org. techn. Rep. Ser., 285, 18 Wolf, H. P., Forster, G. & Leuthrd, F. (1957) Gstroenterologi, 87, 172 Wolfson, S. K., Spencer, J. A., Sterkel, R. L. & Willims- Ashmn, H. G. (1958) Ann. N.Y. Acd. Sci., 75, 26 Young, J. J. (1958) Ann. N. Y. Acd. Sci., 75, 357