A detailed study of the alterations in the serum protein fractions following commencement

of effective anti-tuberculosis treatment was undertaken in 511 patients with

pulmonary tuberculosis. The concentration of serum albumin was 20-30 per

cent higher (P<0.001) and that of a1, a2 and g -globulins significantly lower at

the end of treatment than on admission (P <0.01), the magnitude of decrease being

7-17 per cent for g  -globulin, 19-30 per cent for a1-  globulin and 35-38 per cent for

a2-  globulin. In all, 481 patients had a favourable response to treatment, 46 of

whom had a bacteriological relapse within 18 months of stopping treatment. The

likelihood of a relapse was found to be greater with higher a2  -globulin concentrations

at the end of treatment, the proportions of patients who relapsed being 3, 9,

13 and 15 per cent with a2  -globulin concentrations of < 0.40, 0.40-0.59, 0.60-0.79

and > 0.80 g/dl (P=0.02)

Jayasankar, K

Kailasam, S

Kannapiran, M

Krishnamurthy, M S

Krishnamurthy, P V

Raghupati Sarma, G

NIRT Institutional Repository

Indian J Med Res 81, June 1985, pp 551-557Serum protein profile in patients with pulmonarytuberculosisS. Kailasam, K. Jayasankar, M. Kannapiran, M.S. KrishnamurthyP.V. Krishnamurthy & G. Raghupati SarmaTuberculosis Research Centre (ICMR), MadrasRevised article received February 8, 1985A detailed study of the alterations in the serum protein fractions following commen-cement of effective anti-tuberculosis treatment was undertaken in 511 patients withpulmonary tuberculosis. The concentration of serum albumin was 20-30 percent higher (P<0.001) and that of 1, a2 and g -globulins significantly lower atthe end of treatment than on admission (P<0.01), the magnitude of decrease being7-17 per cent for g -globulin, 19-30 per cent fora1- globulin and 35-38 per cent fora2- globulin. In all, 481 patients had a favourable response to treatment, 46 ofwhom had a bacteriological relapse within 18 months of stopping treatment. Thelikelihood of a relapse was found to be greater with higher a2 - lobulin concentra-tions at the end of treatment, the proportions of patients who relapsed being 3, 9,13 and 15 per cent with a2 -globulin concentrations of < 0.40, 0.40-0.59, 0.60-0.79and >  0.80 g/dl (P=0.02).It has been well established that inchronic infectious diseases like tuberculo-sis, the albumin content of serum proteinsshows a decrease while the globulin contentshows an increase leading to low albuminto globulin (A/G) and albumin to a2 -globulin (A/ a2) ratios1,2. An investigationwas undertaken to study the alterationsin serum protein profile following com-mencement of effective anti-tuberculosistreatment, and to examine whether thesechanges are associated with immediateresponse to treatment and a subsequentlikelihood of relapse.Material & MethodsThe investigations were undertaken in511 patients with sputum-positive pulmo-nary tuberculosis;of these, 422 hadorganisms initially sensitive to isoniazidand streptomycin, and 89 resistant eitherto o e or both the drugs. These patientswere allocated at random to one of thefollowing regimens :R3: Rifampicin (12 mg/kg body weight)plus streptomycin (0.75 g) plus isoniazid(400 mg) plus pyrazinamide (35 mg/kg)daily for 3 months.R5: The same as R3 plus a twice-weeklyphase of 2 months with streptomycin(0.75 g), isoniazid (15 mg/kg) and pyra-zinamide (70 mg/kg).Z5: The same as R5, but without rifam-picin.Blood samples were obtained at 0 and3 months from patients admitted to the551552 Serum proteins in tuberculosisR3 regimen, and at 0, 3 and 5 monthsfrom those admitted to the R5 and 25regimens and also from 18 healthy volun-teers. The total serum protein content wasdetermined by the Biuret method3. T edifferent serum protein fractions wereseparated electrophoretically4 at a pHof 8.6 (0.06M barbitone-sodium barbitonebuffer, µ=0.05) using Whatman No. 3filter paper at a constant voltage of 210 Vfor 5) h. The proteins were stained withmethanolic bromophenol blue, and thedifferent fractions were eluted with 0.01 Msodium hydroxide. The extinction wasrecorded at 540 nm, and the concentrationsof the individual fractions were calculatedon the basis of these values and the totalprotein content of each sample.Counts of viable tubercle bacilliisolated from sputa of patients were setup as described earlier5, and the classi-fication of the radiographic extent oftuberculous disease was undertaken asdetailed earlier6.A patient was deemed to have had anunfavourable bacteriological response ifhe had one or more of 3 cultures positivefor Mycobacterium tuberculosis at each ofthe last 2 months of treatment, irrespectiveof the amount of growth. The responsewas also considered unfavourable in pa-tients who had chemotherapy continuedbeyond the prescribed duration on accountof clinical or radiographic deterioration,and in those who died of tuberculosis. Abacteriological relapse has been defined asthe occurrence of 2 or more positivecultures for M. tuberculosis (of a total of6 cultures) at different months in any 3consecutive monthly examinations, irres-pective of the amount of growth. Treat-ment was started only if at least one of thecultures had a growth of 20 colonies ormore in the presence of at least one posi-tive smear, and also in those who intermit-tently produced positive cultures (of anygrade of growth) over several months.ResultsThe mean values of the serum proteinfractions and the A/G and A/ a2 ratiosin patients with initially drug-sensitiveand drug-resistant organisms before startof treatment, and in healthy volunteersare presented in Table I. The serum albu-min content was lower (about 40%),while the concentrations of all the globulinfractions were higher, ranging from about20 per cent for b -globulin to about 200per cent for a1 -globulin, in the tuberculouspatients than in the healthy volunteersleading to appreciably lower(about60-65%) A/G and A/ a2 ratios in the former.The mean values of all the protein fractionsand the ratios were similar in patientswith initially drug-sensitive and drug-resistant organisms.The association between the concentra-tions of a2 or g -globulin, the A/G orA/ a2 ratios and the disease status onadmission, as measured by the radiogra-phic extent of tuberculous disease andthe log viable count of tubercle bacilliisolated from the sputa of the patients wasexamined (Table II). There was an increa-sing trend in the mean concentrations ofa2 and g -globulins and a correspondingdecreasing trend in the A/G and A/ a2ratios with an increase in the severity ofdisease as measured by either the radio-graphic extent of the disease or the countof viable tubercle bacilli (P<0.01).Changes in the protein fractions duringtreatment are presented in Table III; thefindings in patients with initially drug-Kailasam et al 553Table I. Serum protein concentrations in patients with pulmonary tuberculosis and in healthy volunteers(Data are mean±SD)Serum protein fraction(g/dl)Drug-sensitivepatients (422)Drug-resistantpatients (89)Healthy volunteers(18)Total protein 6.82 ± 0.59Albumin 2.71 ± 0.56a1 -globulin 0.33 ± 0.11a2 -globulin 0.86 ± 0.20b -globulin 0.67 ± 0.14g -globulin 2.24 ± 0.53A/G ratio 0.69 ± 0.25A/ a2 ratio 3.52 ± 2.38Figures in ‘parentheses are numbers studied6.83 ± 0.56 6.64 ± 0.442.78 ± 0.54 4.34 ± 0.350.32 ± 0.11 0.11 ± 0.040.87 ± 0.22 0.48 ± 0.100.66 ± 0.14 0.55 ± 0.042.20 ± 0.53 1.16 ± 0.270.72 ± 0.26 1.93 ± 0.383.53 ± 1.75 9.49 ± 2.20Table II. Association between disease status on admission and serum protein fractions and ratios(Data are mean values)Slight 47 0.73 2.02 1.00 5.36 0 20 0.67 1.91 1.04 6.42Limited 118 0.88 2.09 0.77 3.62 1- 53 0.80 2.19 0.78 3.99Moderate 181 0.90 2.36 0.74 3.68 3- 162 0.84 2.21 0.73 3.58Extensive 161 0.90 2.40 0.63 3.06 5- 217 0.91 2.28 0.67 3.11Gross 4 0.89 2.39 0.60 2.83 7- 53 0.93 2.26 0.60 2.82*Based on 511 patients: **Based on 505 patients onlysensitive and drug-resistant organisms havebeen amalgamated as these were similar.There was little change with treatment inthe total protein content. However, therewas a 20-30 per cent increase in the serumalbumin content during treatment(P<0.001), and significant decreases(P<0.01) in all the globulin fractionsexcepting b -globulin in the 3 groups, themagnitude of decrease being 6-8 per centfor b -globulin, 7-17 percent for g -globulin,19-30 per cent for a1 -globulin and 35-38p  cent for a2 -globulin. Consequently, theA/G and A/ a2 ratios were appreciablyhigher (41-66 and 87-114%, respectively)at the end of treatment in all the groups554 Serum proteins in tuberculosisTable III. Changes in the serum protein concentrations during treatment(Data are mean ± SD)Protein fraction(g/dl)R3 (167) R5 (179)Months of treatmentZ5 (145)0 3 0 3 5 0 3 5Total proteinAlbumina1 -globulina2 -globulinb -globuling -globulinA/G ratioA/ a2 ratio6.93 6.91 6.77 6.78± 0.52 ± 0.54 ± 0.62 ± 0.552.79± 0.530.32± 0.100.86± 0.180.67± 0.142.29± 0.533.35 2.74± 0.46 ± 0.540.26 0.33± 0.12 ± 0.120.56 0.86± 0.14 ± 0.220.62 0.66± 0.16 ± 0.152.12 2.18± 0.50 ± 0.523.35± 0.510.27± 0.150.55± 0.150.59± 0.152.01± 0.480.70 0.99± 0.25 ± 0.293.51 6.57± 1.57 ± 2.440.72 1.04± 0.26 ± 0.343.56 6.68± 2.16 ± 2.696.70± 0.523.51± 0.520.23± 0.110.53± 0.150.61± 0.151.81± 0.461.17± 0.387.39± 2.856.78± 0.567.03± 0.522.68± 0.580.34± 0.110.86± 0.210.66± 0.132.24± 0.533.48± 0.510.25± 0.110.58± 0.150.63± 0.152.08± 0.500.68 1.04± 0.26 ± 0.323.34 6.47± 1.54 ± 2.356.84± 0.513.51± 0.550.26± 0.310.54± 0.130.62± 0.151.91± 0.481.13± 0.377.16± 2.70Figures in parentheses indicate number studied.*Twenty patients (7 R3, 5 R5 and 8 Z5) have beenexcluded due to non-availability of data at all the specified time-points(P<0.001). No association was observedbetween the magnitude of change withtreatment in the protein concentrationsor the A/G or A/ a2 ratio and thedisease status on admission (data notpresented).Of the patients with initially drugsensitive organisms, all but one (R3) had afavourable response; however, a bacterio-logical relapse requiring treatment occurr-ed in 21 (15%) of 136 R3, 4(3%) of 146 R5,and 15 (12%) of 129 Z5 patients within 18months after stopping treatment. Amongpatients with initially drug-resistant orga-nism , response was unfavourable in 1 of30 R3, 3 of 33 R5 and 5 of 16 Z5 patients ;of the remaining 70 patients, 6 (3 R3, 1 R5and 2 Z5) had a bacteriological relapsewithin 18 months after stopping treatment.Since it appears that of all protein fractionsexamined, only the a2 and the g -globulinfractions are likely to be of any importance,further comparisons were made only forthese two fractions and the A/G and theA/ a2 ratios.Kailasam et al 555The mean a2 and g -globulin concentra-tions at the end of treatment in the 10patients (9 of whom had initially drug-resistant organisms) who had an unfavour-able response at the end of thescheduledperiod of treatment were 0.61 and 242g/dl, values higher than in those who had afavourable response (0.54 and 1.94 g/dl,respectively). The mean A/G and A/ a2ratios in the former group (0.86 and 5.85,respectively) were lower than those in thelatter group (1.10 and 7.07 respectively).Comparison of the values in patientswho had a bacteriological relapse withthose who did not (Table IV), showed thatthose who relapsed on the 2 rifampicinregimens had higher a2 -globulin (P<0.01)and g -globulin (P > 0.2) concentrations, andlower A/G (P = 0.04) and A/ a2 (P< 0.01)ratios at the end of treatment than thosewho did not have a bacteriological relapse.For patients in the Z5 regimen, thesevalues were similar in patients who relapsedand in those who did not.Amalgamatingthe results of patients from all regimensat the end of treatment, it was observedthat the mean concentrations of both theglobulin fractions ( a2 and g ) were higher,and the mean ratios (A/G and A/ a2were lower in patients who relapsed thanin those who did not. Thus, the meanvalues for the a2 and g -globulin concen-trations, and the A/G and A/ a2 ratios inpatients who had a bacteriological relapsewere 0.58 and 2.02 g/d1 and 1.04 and 6.28resp ctively; the corresponding values inpatients who did not relapse, were 0.53and 1.93 g/d1 and 1.11 and 7.15 respectively.The differences attained statistical signi-ficance (P<0.04) only with the a2 -globulinconcentration and the A/ a2 ratio. Themean at-globulin concentrations at theend of treatment in patients who relapsedand in those who did not (not tabulated)were 0.28 and 0.25 g/dl, respectively ; thediffer nce was not significant (P>0.2).The likelihood of a relapse was foundto be greater with higher a2 -globulin con-centrations at the end of treatment, theproportions of patients who relapsedTable IV. Serum protein concentration at the end of chemotherapy in patients with bacteriologicalrelapse(Data are mean ± SD)Regimen Group No. of A/G A / a2patientsR3 + R5 Relapse 29 0.61 ± 0.12 2.03 ± 0.50 0.95 ± 0.35 5.71 ± 1.88No relapse 312 0.53 ± 0.15 1.94 ± 0.50 1.10 ± 0.35 7.13 ± 2.71Z5 Relapse 17 0.53 ± 0.14 2.01 ± 0.54 1.18 ± 0.50 7.24 ± 2.78No relapse 123 0.54 ± 0.13 1.89 ± 0.46 1.14 ± 0.35 7.20 ± 2.71All Relapse 46 0.58 ± 0.13 2.02 ± 0.51 1.04 ± 0.42 6.28 ± 2.35regimens No relapse 435 0.53 ± 0..14 1.93 ± 0.49 1.11 ± 0.35 7.15 ± 2.71556 Serum proteins in tuberculosisbeing 3, 9, 13 and 15 per cent with a2-globulin concentrations of <0.40, 0.40-0.59, 0.60-0.79 and > 0.80 g/dl, respectively,a significant trend (P=0.02). Such a trendwas not observed with the other globulinfractions.Of the 46 patients who relapsed within18 months after stopping treatment,32 had an early relapse (within 6 months),and the remaining had a late relapse(7-18 months). The mean a2 -globulin con-centrations in the early and late relapseswere 0.61 and 0.53 g/dl, respectively(P=0.06); the mean A/ a2 ratios in the2 groups were 5.99 and 6.93, respectively(P>0.2). The mean r-globulin concen-trations and A/G ratios (results notpresented) were similar in the 2 groups.DiscussionThe study of serum protein profilebefore and during treatment with shortcourse regimens was undertaken primarilyto identify factors of prognostic significancein patients with pulmonary tuberculosis.Weak associations, though statisticallysignificant, were observed between theextent of tuberculous disease on admissionand the concentrations of a2 and g -glo-bulins and the A/G and A/ a2 ratios.During treatment, the mean decrease inthe concentration of a2 -globulin was morepronounced than that of the other globu-lins, and the increase in the A/ a2 ratiowas substantially higher than that of theA/G ratio. Similar observations weremade by Gilliland and others1 who sug-gested that the A/ a2 ratio could be emplo-yed to assess the activity of the diseaseand to monitor the progress of the patientduring chemotherapy.Almost all the patients with initiallydrug sensitive organisms, and a substantialproportion of those with organisms initiallyresistant tostreptomycin or isoniazidor both had a bacteriologically favour-able response at the end of the scheduledperiods of chemotherapy with effectiveshort-course regimens containing rifam-picin and pyrazinamide in addition toisoniazid and streptomycin; however, bac-teriological relapses requiring treatmentoccurred in varying proportions ofpati nts with the 3 regimens investigatedafter stopping treatment. Results reportedin this paper suggest that of all the proteinfractions examined, the a2 -globulin fra-tion is probably of greater prognosticsignificance than the other fractions.The classification of the proportions ofrelapses according to the a2 -globulin con-centrations at the end of treatment showedan increase with increasing concentrations;it was, however, not possible to determinethe level of the concentration at whichthe outcome of a relapse could be pre-dicted. The concentration of this fractionis known to be elevated in conditionswhere tissue necrotic changes are knownto occur. The observation that the 2 -globulin concentration was higher inpat e ts who relapsed than in those whodid not suggests that active tissue des-truction is possibly still in progress inth former. The mean concentration ofthis protein fraction was also slightlyhigher in patients who relapsed early(within 6 months after stopping treatment)than in those who had a later relapse(7-18 months) suggesting a greater degreeof active tissue destruction in the earlyrelapses.The a2 -globulin fraction consists ofa number of proteins with similar electro-Kailasam et al 557phoretic mobilities, some of which likeceruloplasmin are acute phase proteins.The concentrations of these proteins areknown to be increased in response toinflammatory reactions involving tissuedamage and infection7 The elevated con-centrations of the a2 -fraction in tubercu-lous patients before start of treatment,and the slightly higher concentrations inpatients who had an unfavourable responseor in those who had a bacteriologicalrelapse after stopping treatment mightbe due to the higher concentrations ofthese acute phase proteins. However, nofirm conclusions on the possible signi-ficance of the findings reported here canbe drawn until data are available on thea2 -globulin and the constituent acutephase protein concentrations at time-pointsbetween the end of treatment and theoccurrence of relapse.AcknowledgmentThe authors are grateful to the medical andnursing staff of the Centre for organizing thecollection of blood specimens, and to Shri V.Sundaram for computational assistance.References1. Gilliland, I.C., Johnston, R.N., Stradling, P.and Abdel-Wahab, E.M. Serum proteins inpulmonary tuberculosis. Br Med J 1 (1956)1460.2.3.4.5.6.7.Gilliland, I.C., Stradling, P. and Abdel-Wahab, E.M. Serum protein changes follow-i g BCG vaccination. Br Med J 1 (1958)87.White, W.L. and Frankel, S. Seiverd’s chemistryfor medical technologists, 2nd ed. (The C.V.Mosby Co., Saint Louis) 1965 p 147.Block, R.J., Durrum, E.L. and Zweig, G.A manual of paper chromatography and paperelectrophoresis, 2nd ed. (Academic Press,Inc., New York) 1974 p 554.Tuberculosis Chemotherapy Centre, Madras.A controlled comparison of two fully super-vised once-weekly regimens in the treatmentof newly diagnosed pulmonary tuberculosis.Tubercle 54 (1973) 23.Tuberculosis Chemotherapy Centre, Madras.A concurrent comparison of isoniazid plusPAS with three regimens of isoniazid alonein the domiciliary treatment of pulmonarytuberculosis in South India. Bull WHO 23(1960) 535.Kushner, I. The phenomenon of acute phaseresponse. Ann NY Acad Sci 389 (1982) 39.Reprint requests :The Director, Tuberculosis Research Centre, Spur Tank RoadChetput, Madras 600031

Serum protein profile in patients with pulmonary

tuberculosis

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Serum protein profile in patients with pulmonary tuberculosis

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