Tubercle (1990) 71, 193-197 @ Longman Group UK Ltd 1990

Changes in serum proteins (albumin, immunoglobulins and acute phase proteins) in pulmonary tuberculosis during therapy

C. T. WONG* and N. SAHAt

*Department of Physiology and tDepartment of Paediatrics, Faculty of Medicine, National University of Singapore, Republic of Singapore

Summary-Serum levels of three immunoglobulins (IgG, IgA & IgM), albumin and six acute phase proteins (al-antitrypsin, haptoglobin, transferrin, a2-macroglobulin, complements C3 & C4) were measured by immunoelectrophoresis in 57 patients with pulmonary tuberculosis when they were first diagnosed, and followed at 1 month, 2 month and 4 month intervals during anti-tuberculosis treatment. These values were compared to those from 41 healthy controls. Significant increases were observed in the initial values of serum IgG, IgM, al- antitrypsin and haptoglobin, while transferrin and a2-macroglobulin levels were significantly reduced. No changes were observed in the levels of serum albumin, IgA and complement C3. Levels of all measured proteins decreased with treatment. However, at 4 months, IgG and al-antitrypsin were still significantly elevated when compared to control values, while the level of haptoglobin had decreased to a level significantly lower than that of the control value.

Introduction

It is well established that changes in levels of serum proteins occur in response to both acute and chronic infections. This is especially true of immunoglobulins and acute phase proteins. In an infection like tuberculosis, such changes in serum protein levels are expected. In a survey of the literature, a number of reports on inconsistent changes in the levels of various serum proteins in pulmonary tuberculosis were found [l-9]. Serum

*Correspondence to: Dr C. T. Wong, Department of Physiol- ogy, Faculty of Medicine, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511, Republic of Singapore.

IgG and IgA levels have been mostly reported to be increased, while most of the authors reported unchanged IgM levels. Only three papers had been published on the levels of acute phase proteins in pulmonary tuberculosis. Grange et al. [6] reported significant increases in circulating levels of al-antitrypsin, a2-macroglobulin, hap- toglobin and complement C3 and a significant decrease in transferrin levels while Emmett et al. [S] reported a significant increase only in serum al-antitrypsin level. A recent report confirmed the findings of Grange et al. [6] with the exception of a2-macroglobulin, which was decreased [9]. IgG and IgA were increased while IgM was decreased in this report. Recently we have re-

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ported significant increases in serum IgG, IgM, al-antitrypsin and haptoglobin levels in pulmon- ary tuberculosis, while serum transferrin and a2-macroglobulin levels were decreased [lo].

However, it is not clear if the changes in serum protein concentrations have any aetiological signi- ficance or prognostic value. To explore this further, we report here the results of a serial study of 57 patients whose serum protein concentrations were measured on diagnosis and during the course of treatment.

Materials and methods

The sample population comprised 57 patients (39 males & 18 females), in the age range 16-74 years, all confirmed to be suffering from pulmonary tuberculosis by positive sputum culture and radiol- ogical evidence. All patients were treated with a combination of four drugs per day for 2 months (rifampicin, 45&600 mg; isoniazid, 300 mg; pyra- zinamide, 1.5-2.0 g; ethambutol, 25 mg/Kg body weight or 800 mg) followed by a combination of two drugs per day for 4 months (rifampicin, 450- 600 mg; isoniazid, 300 mg). All the patients in the series recovered and were certified as medically fit by 4 months. They also completed the full course of treatment for the 6 months. On diag- nosis, a blood sample was obtained. A further three samples were obtained from the same pati- ents at the 1st 2nd and 4th month after the start of medication. Serum was separated and stored at -20°C until analyzed. A total of 41 healthy Chinese male blood donors, aged from 24-37 years, formed the control series.

Serum protein concentrations were measured by rocket immunoelectrophoresis in agarose gels [ll]. 1.57 g o a arose gels (Calbiochem-Behring, type A) in barbital buffer (pH 8.6) containing 1.0% anti-human antibody to the respective pro- tein were used. 5.0 ul of appropriately diluted samples and standards of known protein con- centrations (Behringwerke AG) were placed in wells 2.5 mm in diameter. The electrophoresis was run with 10 V/cm for 4 h followed by 20 V/cm for 0.5 h at 4°C. The gels were washed overnight in 0.9% NaCl, dried, and stained in 0.5% Coomasie brilliant blue R (Sigma Chemical Co.). After staining, the heights of the rockets were measured. The protein concentrations of the samples were calculated from a standard curve produced by the heights of the rockets of known protein standards on the same electrophoresis plate.

Results and discussion

Since the samples were of mixed sexes, the effect of sex on the concentrations of the different serum proteins were examined. IgM concentration was found to be significantly higher (P < 0.05) in females (1.48 f 0.55 g/l) than in males (1.08 + 0.40 g/l). Further analysis of the results showed that this difference persisted throughout the whole observation period, though it was signi- ficant only at 0 months and 1 month and is consis- tent with previously reported observations in healthy populations [l, 12, 131.

The effects of age on the protein concentrations of the samples were also investigated at 0 months since the age range of the patients were quite wide while that of the controls were from a much younger population. The patient samples were divided into two groups, namely a younger group (below 40 years) and an older group (above 40 years). No significant difference in any of the serum protein concentration was observed between these two groups in both males and females.

Cross-sectional study Serum protein concentrations of patients were compared to control values in Table 1 and Figure 1. No significant difference was observed for albumin levels of patients at 0 months and controls.

IgG was significantly higher (P < 0.001) at 0 months compared to the control population while no significant difference was observed in their IgA levels. IgM levels of male patients at 0 months was also significantly higher (P < 0.01) when compared to the control values. In general, IgG levels have been reported to be increased in pulmonary tuberculosis [l-7, 91 while Bhatnagar et al. [14] reported no significant change. IgA levels have been reported to be increased in several studies [l, 3-6, 9, 141 while unchanged in one [2]. IgM levels have also been reported to be increased in two studies [3,6] while several studies revealed no significant change [l, 2, 4, 5, 7, 141.

Of the acute phase proteins measured, al- antitrypsin and haptoglobin concentrations were significantly increased in the patients (P < 0.001 and P < 0.01, respectively). However, transferrin and a2-macroglobulin were decreased significantly (P < 0.001) while no change was observed for complement C3. The changes in serum concentra-

SERUM PROTEINS IN TUBERCULOSIS 195

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tions of al-antitrypsin, haptoglobin and transfer- rin in tuberculosis were similar to the observations of Grange et al. [6]. However, both a2-macroglo- bulin and complement C3 were also found to be increased in their series. Caplin et al. [9] reported significant increases in al-antitrypsin, ceruloplas- min and haptoglobin values while a2-macroglobu- lin was decreased and no change was observed for transferrin concentration. The increased al- antitrypsin and haptoglobin levels in pulmonary tuberculosis may be related to their physiological functions. Haptoglobin may restrict utilization of released haemoglobin by pathogenic bacteria by binding to it while al-antitrypsin, with its anti- proteolytic activity, may limit the damage caused by proteinases released from granulocytes in- volved in the inflammatory response. An increase in hepatic synthesis of these proteins is believed to be the cause of the rise in their plasma levels [ 151. A fall in transferrin concentration is difficult to explain. During the inflammatory response, there may be an increase in iron uptake by the cells due to demand for microbial growth. An important function of a2-macroglobulin is the inhibition and elimination of endopeptidases released extra- and intra-vascularly. The complexes are normally removed from the blood within an hour. This may lead to a decreased serum concentration of this protein observed in the pulmonary tuberculosis.

Serial study The concentration of all the measured serum proteins in the patients decreased with treatment (Table 1 & Fig. 1). Albumin concentrations de- creased significantly by 4 months from that of control values (P < 0.01). However, no significant difference was observed during the course of treatment. A decrease in serum albumin level normally reflects protein malnutrition [16]. The slight decrease observed in these patients may be due to a decrease in food intake with the progression of the disease or to the side-effects of the medication. Since they were outpatients, we could not determine whether there was a change in their dietary habits after the diagnosis of tuber- culosis.

IgG concentration was significantly lower at 4 months than that at 0 months, but was still significantly higher than that in the control series. IgM concentration at 4 months was not signifi- cantly different from 0 months or control values.

WONG AND SAHA

The general trend is that, as the condition of the patients improved, the levels of all three immunoglobulins decreased towards control values. IgA concentration remained stable at 1 month, but decreased slightly at 2 months and was significantly lowered (P < 0.05) only at 4 months compared to that at 0 months.

al-antitrypsin concentration at 4 months had decreased significantly (P < 0.01) with treatment when compared to 0 months but it was still significantly higher than control values (P < 0.001). At 4 months haptoglobin concentra- tion was also significantly reduced compared to both 0 months and control values. This probably reflects an increased catabolism of haptoglobin as the haptoglobin-hemoglobin complex is cleared from the circulation by the liver and suggests a regulatory function of this protein. Transferrin and aZmacroglobulin concentrations were further reduced with treatment. Transferrin level at 4 months was significantly lower than at 0 months (P < 0.001). While no significant change was observed for complement C3, complement C4 was marginally reduced (P < 0.05) after treatment.

In conclusion, significant changes in the levels of several serum proteins have been observed to be associated with tuberculosis. Though they may not be of diagnostic value, they do, however, indicate the response of the body to the infection and recovery after treatment. In particular, the decreasing levels of IgG and IgM with treatment may reflect response to medication in the patients.

Acknowledgements

The authors would like to thank the Medical Director of the Singapore Anti-Tuberculosis Association (SATA) Chest and Heart Clinic for permission to carry out this study. This work was supported by grants from the Singapore Turf Club and Singapore Anti-Tuberculosis Association.

References

Faulkner JB, Carpenter RL, Patnode RA. Serum protein and immunoglobulin levels in tuberculosis. Am J Ch Patholl967; 48: 556-560. Buckley CE, Dorsey FC. A comparison of serum immu- noglobulin concentrations in sarcoidosis and tuberculosis. Ann Int Med 1970; 72: 3742. Alarcon-Segovia D, Fishbein E. Serum immunoglobulins in pulmonary tuberculosis. Chest 1971; 60: 133-136. Grange JM, Gibson J, Nassau E, Kardjito T. Enzyme- linked immunosorbent assay (ELIZA): a study of anti- bodies to Mycobacterium tuberculosis in the IgG, IgA and IgM classes in tuberculosis, sarcoidosis and Crohn’s disease. Tube&e 1980; 61: 145-152.

SERUM PROTEINS IN TUBERCULOSIS 197

Table 1 Serum protein concentrations of control and patient samples at 0, 1,2 and 4 months after commencement of anti-tuberculosis treatment. (Mean f SD; g/l)

Protein

AIb IgG IgA IgM Tf alAt o2Mg c3c HP C4

Control (n = 41) Patients (n = 57) 0 months I month 2 months 4 months

43.0 f 3.41 40.1 f 11.0 39.6 f 9.62 38.0 + 9.53 36.4 f 9.81c’ 10.5 f 3.70 19.7 + 6.34” 18.2 f 6.08 16.2 f 6.52 14.6 f 6 01b2@ 3.70 f 1.12 3.90 + 1.47 3.92 f 1.51 3.36 f 1.53 3.04 + 1:3ob3 0.75 f 0.29 *1.20 f 0.49sz *1.21 f 0.54 ‘1.07 + 0.53 *0.95 + 0.45 2.86 + 0.38 2.32 + 0.62” 2.26 f 0.65 1.99 + 0.63 1.82 + 0.46b1~c1 1.63 f 0.49 4.47 + 1 66a’

2.01 + o:wi 4.51 + 1.67 3.85 f 1.44 3.47 + 1.23b2”’

2.71 + 0.62 2.08 + 0.78 1.84 + 0.73 1 67 f 0.56” ’ 0.88 + 0.20 0.89 f 0.25 0.89 f 0.30 0.76 + 0.22 0.75 + 0.30

2.18 f 0.89 3.40 f 1.77” 2.38 + 1.32 1.86 + 1.14 1.60 f 1.05b’s” nd 0.30 f 0.13 0.34 * 0.21 0.25 f 0.11 0.23 f 0.13b3

5.

6.

7.

8.

9.

10.

*Values for 39 male samples only. Control versus 0 m al = P < 0.001

a2 = P < 0.01 Omversus4mbl= P<O.oOl

b2 = P < 0.01 b3 = P c 0.05

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11.

12.

13.

14.

15.

16.

Control versus 4 m cl = P < 0.001 c2 = P < 0.01 c3 = P < 0.05

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