PLASMA COPPER AND ZINC LEVELS IN PULMONARY TUBERCULOSIS

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1 PLASMA COPPER AND ZINC LEVELS IN PULMONARY TUBERCULOSIS B.K. KHANNA, R. KUMAR, P.K. MUKERJI,* A.R. CHOWDHURY AND V,P. KAMBOJ** Summary : A study of plasma copper and zinc level was conducted in 46 normal subjects and 94 patients of pulmonary tuberculosis. 64 patients had received specific anti-tuberculosis therapy for over one month and 30 were previously untreated. Plasma copper and zinc levels were recorded initially in all patients and subsequently after 1, 2 & 3 months chemotherapy in the latter group ouly. Itwas found that pulmonary tuberculosis is characterised by rise in level of plasma copper and decline in plasma zinc level. Only 14 patients did not register any alteration in their levels Highly significant correlation was established between the level of these trace elements and the presence of acid-fast bacilli in the sputum, extent of pulmonary involvement, and the nature of lesions seen on chest X-ray. Clinical, bacteriological and radiological improvement following effective therapy led to partial reversal of these changes. The depression of plasma zinc level was found to be more pronounced and a highly sensitive index of tuberculous activity compared to elevation of plasma copper level. Introduction Elevation of plasma copper and/or depression of plasma zinc level (Tani 1965, Halsted and Smith 1970, Underwood 1971, Beisel, Pekarek and Wannemacher 1974, Bogden et al 1977) has been reported in pulmonary tuberculosis and in many other chronic diseases. Most of the studies on these two biochemical parameters relate only to one of them. For example, whereas Tani (1965) has emphasised the importance of measuring serum copper as a useful biochemical modality in pulmonary tuberculosis, Halsted and Smith (1970) have demonstrated reduced plasma zinc level. Bogden et al (1977) have drawn attention to the importance of measuring both the parameters simultaneously in these cases, although in most of their cases, high copper concentration and low zinc level was not encountered simultaneously. Pulmonary tuberculosis has a wide spectrum of presentation. The lesions, radiographically, may range from acute pneumonic form to that of chronic fibroid lesion. Likewise, the symptoms may also vary from severe to none. It is well recognised that many patients, especially those following chemotherapeutic treatment, may not excrete tubercle bacilli in their sputum, though they continue to suffer from active pulmonary tuberculosis. Consequently, it is expected that the basic metabolic alteration, encountered in this disease, will vary according to the mode of presentation, severity of the disease and presence or absence of tubercle bacilli in the sputum. Adaptation of a single criterion for the diagnosis of pulmonary tuberculosis e.g. isolation of T.B. from the sputum, will necessarily restrict the spectrum of case studies (Bogden et al 1977). Furthermore, the authors felt that correlation of these biochemical parameters, with the clinical, radiographic and bacteriological status of the patient may be essential. Further, an effort has been made in this study, to record the effect of anti-tuberculosis chemotherapy, administered for 3 months, on plasma copper and zinc concentration. Material and Methods The material for the study was selected from human volunteers (controls) and from proved patients of pulmonary tuberculosis admitted to the indoor wards of the department of tuberculosis and respiratory diseases. The control group included doctors, nurses and other staff members working in the department. They were thoroughly examined and had a negative medical history. They were not receiving any medication (46 subjects). 94 patients of pulmonary tuberculosis were included in the study. 46 patients were sputum positive (by smear examination) at the time of admission to the study, while 48 patients were reported to have been sputum positive in the past, though negative at the time of the study. Patients who had hepatic or renal disorders, diabetes mellitus, hypertension, rheumatoid disease, lymphoma, myocardial infarction, pernicious anaemia, hyperthyroidism and chronic alcoholics were excluded. Women who were pregnant or were taking oral contraceptives were also not included. Detailed history, particularly relating to previous therapy, was taken. On the basis of history of previous anti-tuberculosis treatment the patients were divided in two groups: (a) The random group: Patients who had *Department of Tuberculosis, K.G s Medical College, Lucknow (India). **Division of Endocrinology, Central Drug Research Institute, Lucknow

2 180 B.K. KHANNA, et al., had anti-tuberculosis therapy for more than a month prior to their induction in the study (64 cases). (b) The follow up group: Patients who had not been treated previously, or who had taken less than one month of antituberculosis chemotherapy. After initial observations, these patients were put on specific anti-tuberculosis therapy; comprising of either streptomycin 1 gm. I.M.I, once a day. I.N.H. 300 mg. orally once a day and PAS 10 gms. orally (S-f P+H in 6 patients) or streptomycin and I.N.H. in the same dose as above and thioacetazone 150 mg./day orally in place of PAS (S+T+H in 21 patients) or streptomycin + I.N.H. in the same dose as above and Ethambutol 1000 mg. by month/day (S4- H+E in 2 patients) or streptomycin + I.N.H. in the same dosage as given above (S+H in 1 patient).for a period of 3 months. All the clinical and biochemical observations were recorded in these patients at the end of 1, 2 and 3 months of therapy (30 patients). Observations recorded in these patients included detailed clinical examination, routine laboratory investigations including sputum smear examination for A.F.B. and special investigations such as plasma copper and zinc estimation. The method for assessment of plasma copper and zinc was the same as that employed by Bogden et al (1977). The blood samples were withdrawn between the hours of 10 A.M. and 4 P.M. which coincided with the working hours of the department. Observations A. Correlation between plasma copper and plasma zinc levels in tuberculosis patients and control subjects: Correlation between plasma (Cu) and Zinc (Zn) in relation to normal values at the time of admission to the study. A significant rise of 22% in plasma copper level (154.5 ug% ± 33.6) took place in the patients in contrast to controls (t = 6.04, df = 138, P < 0.001), while the decline in the plasma zinc level (63.6 ug% ±17.8) was 28.8%. The difference was highly significant (t = 10.22, df = 138, P < 0.001). The reduction in zinc level was more pronounced. However, no significant difference was observed in plasma copper and plasma zinc level between treated and untreated patients (t = 1.24, df = 42, P > 0.05). Out of 94 patients, there were 69 males and 25 females. Their age varied from 10 years to 50 years. No statistical correlation could be obtained between plasma copper and plasma zinc values with relation to age and sex of the patients. The dietary level of copper and zinc in this study was not estimated, though all the patients continued to receive the standard hospital diet. B. Copper/Zinc ration in tuberculosis patients and control subjects: Copper/Zinc ratio was estimated in 46 controls and 94 tuberculosis patients. The ratio was 2.61 ± 0.85 in tuberculosis cases, while it was 1.44 ± 0.27 in controls. The difference was highly significant (t= 12.14, df= 138, P < 0.001). 80 out of 94 patients showed abnormalities of either plasma copper level or of plasma zinc level or both. 14 patients had normal plasma copper as well as zinc level. 20 patients had normal plasma copper and low plasma zinc level (value less than 70 ug%). There were 6 patients who had high plasma copper level (value exceeding 145 ug%) and normal zinc TABLE 1 Correlation Between Plasma (Cu) and Zine, (Zn) in Relation to Normal Values at the time of admission to the study Ind. J, Tub., Vol. XXIX, No. 3

3 PLASMA COPPER AND ZINC LEVELS IN PULMONARY TUBERCULOSIS 181 level, while 54 patients had high plasma copper As there was no statistical difference in level and as well as low plasma zinc level, plasma copper and zinc concentration between treated and untreated groups (vide table 1), C. Correlation between plasma copper con a11 the Patients (94) were clubbed together for centration and zinc concentration with sputum analysis. status of tuberculosis patients: TABLE 2 Correlation between Plasma Copper and Zinc Levels with Sputum Status Sputum for A.F.B. No. of Ps. Plasma Copper Plasma Zinc Meam (ug%) ± S.D. % rise Meantug%) ± S.D. % fall Positive ± ± Negative ± ± Normal Value: 126.6ug% ± ug% ± 9. 5 It is apparent from table 2 that plasma copper concentration was significantly higher in sputum positive cases (t=5.61 df=92, P<0.001) while plasma zinc showed significantly lower level in the same group (t=4.21, df=92, P < 0.001). However, change in plasma zinc was more pronounced in bacillary cases as compared to the copper level. D. Correlation between plasma copper concentration zing concentration with radiological finding Plasma copper concentration rose with the increasing parenchymal involvement. The difference of plasma copper level between the three groups was highly significant (between I & II t=4.78, df=75, P < 0.001, between II & HI 1= 6.45, df=64, P < and I & III t=ll, df=43 P < 0.00n. Similarly plasma zinc concentration decreased with increasing extent of parenchymal involvement. The difference was highly significant between the groups I & III and groups II & III. However, the difference between groups I & II was not statis- TABLE 3 CORRELATION Of Plasma Copper and Zinc Level WITH Radiological Extent Of Disease Group No. of lung No. of Pts. Plasma Copper Plasma Zinc 7ones involved Mean (ug%) ± S.D. % rise Mean (ug%) ± S.D. % fall One ± ± I II III Two ± ± Three ± ± Four ± ± Five ± ± Six ± ± Normal Value ug% ± ug% ± 9.8

4 182 B.K. KHANNA, et at., tically significant (between I & II 1 = 1.77, patients who had improved with the therapy, df=75, P > 0.05; II & III 1=3.52, df=64, irrespective of the therapy administered. Con- P < 0.001; and I & III t==3.86, df=43, P < sequently, the values obtained in these patients 0.001). Plasma zinc changes were manifest even are being presented below as a single group, in patients with one or two zones involvement as compared to serum copper level which In the patients, who showed improvement showed no change from normal in these patients due to chemotherapy, plasma copper level (i.e. those with less than 3 zones involvement). declined from mean value of ug% to TABLE 4 Table showing the levels of Plasma Copper and Zinc in Relation to Predominant Nature of the Lesion Nature of predominant lesion No. of patients Plasma Copper Plasma Zinc Mean (ug%) ± S.D. % rise Mean (ug%) ± S.D. % fall Exudative ± ± Productive ± ± Cavitary ± ± Fibrotic ± Miliary Normal Value : ug% ± ug% ±9.8 Plasma copper concentration was highest in cases suffering from miliary tuberculosis. It showed a rise of 51.6% from normal value. Plasma zinc level was lowest in cavitary cases and registered a fall of 37 % from the normal. We were unable to find any correlation of plasma copper and zinc level with duration of disease as reported by the patient (which, no doubt, was highly subjective), mean temperature of the patients, and the average size of the cavities seen in our cases. E. Follow up group: Out of a total 94 patients, only 30 patients stayed in the wards for a period of 3 months to permit us a follow up of these patients. They were administered (S+P+H in 6 cases, S+Tf-H in 21 cases, S+H+E in 2 cases and S-j-H in 1 case) four different regimen. 25 of them improved clinically, radiographically and bacteriologically. On the other hand, 5 of them had remained stationary. There was no different in plasma copper and zinc levels between the various sub-groups of ug%, but the difference was not statistically significant (t=0.43, df=48, P > 0.05). The level of plasma zinc rose with effective chemotherapy. The rise was found to be significant (t== 3.10, df=48, P < 0.01). On the other hand, cases showing no improvement did not have any significant effect on the plasma zinc level. Discussion Pulmonary tuberculosis affects metabolism of trace elements like any other infectious disease. High plasma copper and low plasma zinc concentrations obtained in our cases reflect only one aspect of these disturbances. Similar observations have been recorded by other workers too (Tani 1965, Halsted and Smith 1970, Underwood 1971, Beisel, Pekarek, Wannemacher 1974, Bogden et al 1977), though some of them have highlighted the alterations in plasma copper level (Tani 1965) while others have focused attention on the plasma zinc level (Halsted and Smith 1970). Bogden et al (1977) have attempted to study both the trace metals at the same time in proved cases of pulmonary tuberculosis and have come to similar con-

5 PLASMA COPPER AND ZINC LEVELS IN PULMONARY TUBERCULOSIS 183 TABLE 5 Correlation of Plasma Copper ami Zinc Level with Response to Therapy in Follow-up group (30 Patients) No. of Patients Before treatment Alter 3 months treatment Plasma Cu Plasma Zn Plasma Cu Plasma Zn Mean (ug%) ± S.D. % Mean rise (ug %) ± S.D. % Mean % fall (ug%) change ±S.D. Mean (ug%) ± S.D. % change Improvement ±25 No Improvement ± ± ± ±23 (fall) ±8.8 (rise) 78 ± ±34 13 (rise) 2.9 (fain elusions. However, they have raised and left many questions unanswered, which no doubt, will have serious bearing on the trace element metabolism in pulmonary tuberculosis. In this study we have attempted to answer some of these. Plasma Copper and Zinc Tuberculosis levels in Pulmonary Rise in plasma copper and fall in plasma zinc level was recorded in our cases. Both of these alterations were found to be statistically significant. Fall in plasma zinc level was more pronounced compared to rise in plasma copper level. Lowering of plasma zinc has been considered to be a more sensitive index of tuberculous infection compared to rise in plasma copper level (Beisel, Pekarek and Wannemacher 1974). Consequently, Bogden et al (1977) concluded that low plasma zinc level with normal plasma copper may mean that infection is in early stages. Patients of pulmonary tuberculosis, who had demonstrable acid fast bacilli (A.F.B.) in sputum smear examination had higher plasma copper level and lower plasma zinc level compared to those who did not have A.F.B. in their sputum smear. This again shows that the activity of the lesion has a direct bearing on the plasma level of these trace elements. The same was found to be true for patients who had lesions extending beyond two zones of the lung. Lesionwise, patients with miliary tuberculosis had highest rise in plasma copper level and maximum depression of plasma zinc level. These cases were closely followed by patients having cavities in the lungs. The patients having predominantly productive lesions had minimal alterations in plasma level of these trace elements. Those having predominantly fibrotic lesion did not have any alteration in their plasma copper level, though slight depression in their zinc level was seen. It might, therefore, seem that alterations in the plasma level of these trace elements can be directly associated with evidence of activity of tuberculous lesions which may be direct, for example, sputum containing acid fast bacilli or indirect such as nature and extent of pulmonary radiographic lesion. Understandably, because of the highly subjective and variable factors, no clear cut correlation could be obtained between plasma level of these trace elements and age and sex of patients, duration of illness, temperature of patients and size of the cavities as observed radiographically. Alterations noted after 3 months of effective chemotherapy The patients who had improved following therapy revealed elevation of plasma zinc level which was more pronounced and more rapid

6 184 B.K. KHANNA, et al., compared to depression in plasma copper level. Rise in plasma zinc level was found to be significant though decline in plasma copper level was not statistically significant. Plasma zinc level, therefore, appears to be a more sensitive indicator of activity of the tuberculous lesion as compared to plasma copper level. In patients who did not improve with chemotherapy, the process of rise in the plasma copper and decline in plasma zinc continued unabated. Four schedules of chemotherapy were administered in our patients. Alterations in the level of these trace elements did not appear to be influenced by the chemotherapy employed. Indeed it was the improvement in clinical condition of the patients which appeared to be the only influencing factor. The more gradual change in the plasma copper level is consistent with the concept that a period of time is required to allow the liver to synthesize and release the copper binding protein, ceruloplasmin (Beisel, Pekarek and Wannemacker 1974). The persistence of high copper concentrations in contrast to more rapid return of zinc concentration to base line value could be because of relatively long half life of ceruloplasmin (Beisel, Pekarek and Wannemacker 1974). Tani (1965) has demonstrated that serial measurements of both serum iron and copper concentrations could not only serve as an indicator of a favourable host response to chemotherapy but also as an early sign of relapse. Our study has confirmed that serial measurements of plasma copper and, more importantly, plasma zinc level may serve as an indirect pointer to the diagnosis of tuberculosis, to the degree of tuberculo-toxaemia, to the extent and nature of the pulmonary involvement and to the favourable response of the host to the specific anti tuberculosis therapy. The role of kidneys in retention of copper and increased excretion of zinc leading to alterations in the serum level of those trace elements has not yet been defined. REFERENCES 1. Beisel W.R., Pekarek R.S., Wannemacker R.W., : The impact of infections disease on trace element metabolism of the host. Trace element metabolism in animals. Volume 2. Edited by Hoekstra W.G., Suttie, J.W., Ganther, H.E. et al. Baltimore, University Park Press, 1974, pp Bogden, J.D., Lintz, D.I., Joselow, M.M., Charles, J., and Salaki, J.S. Effect of pulmonary tuber culosis on blood concentrations of copper and zinc, Am. J. Clin. Path.; 1977, 67, Halsted, J.A., and Smith J.C. Plasma zinc in health and diseases, Lancet; 1970, Tani P. Serum iron, copper, iron-binding capacity and marrow hemosiderin in pulmonary tuberculosis, Ann. Med. Intern. Fenn.; 1965, 54 Suppl. 44, Underwood, F.J. (1971) Trace elements in human and animal nutrition, New York Academic Press, 1971, pp , pp