INTERNATIONAL JOURNAL OF LEPROSY^ Volume 59, Number I

Printed in the U.S.A.

Serum Zinc/Copper Ratio in Subtypes of Leprosy andEffect of Oral Zinc Therapy on Reactional States'

Joseph George, V. N. Bhatia, S. Balakrishnan, and G. Ramu 2

The role of trace elements such as zincand copper in a chronic infectious disease,such as leprosy, has been well understoodin recent years. Zinc is a vital element in anumber of enzyme systems ( 5 ' ' 6 ' ' 8). In hu-man beings, zinc deficiency results in thy-mus atrophy ( 6) and impaired cell-mediatedimmune (CMI) reactions (I. 12. 14 ) . Zinc de-ficiency in various pathological conditionsmanifested by malnutrition was first ob-served by Prasad and co-workers 20). Sig-nificant decreases in serum zinc levels in alltypes of leprosy, except tuberculoid, havebeen reported (II , 22

) . A close association be-tween chronic skin ulceration and loweredserum and skin zinc concentrations has beennoticed in leprosy ( 7 ' ' 3 ' 23). Copper plays animportant role in a number of metalloen-zymes, including cytochrome c oxidase, su-peroxide dismutase, and lysyl oxidase

I7,25 , .) An increase in serum copper inacute and chronic infections has been re-ported ( 2). Ramu and Nagarajan ( 2 ') haveobserved an increased concentration of se-rum copper in reactional states of lepro-matous leprosy. Venkatesan, et a!. (30) havenoticed hypercupremia along with losweredserum zinc levels in active lepromatous lep-rosy and in leprosy patients in reactionalstates.

In the present study, serum zinc and cop-per levels and serum zinc/copper ratios wereestimated in a large number of subjects who

' Received for publication on 22 February 1990; ac-cepted for publication in revised form on 23 August1990.

J. George, M.Sc., Research Officer, Department ofBiochemistry; V. N. 13hatia, M.D., Director (Micro-biology), Central Leprosy Teaching and Research In-stitute, Chengalpattu, Tamil Nadu 603 001, India. S.Balakrishnan, M. Sc., Ph.D., Head, Department of Bio-chemistry; G. Ramu, M.D., Senior Physician, SacredHeart Leprosy Centre, Kumbakonam, Tamil Nadu 612401, India.

Reprint request to Dr. Ramu.Present address for Joseph George, M. Sc.: Depart-

ment of Biochemistry, Central Leather Research In-stitute, Adyar, Madras 600 020, India.

included leprosy patients across the spec-trum of the disease, patients with lepra re-action with and without oral zinc therapy,and healthy controls.

MATERIALS AND METHODSA total of 225 leprosy patients (both out-

patients and inpatients) and 86 healthy con-trols were included in the study. Out of the225 leprosy patients, 45 were borderline tu-berculoid (BT), 31 were borderline lepro-matous (BL), and 117 were lepromalous(LL). The remaining subjects of the studywere 16 LL cases with erythema nodosumleprosum (ENL) reaction and another 16cases with ENL reaction receiving oral zinctherapy.

The staff members and trainees of the in-stitutions (Central Leprosy Teaching andResearch Institute, Chengalpattu, and Sa-cred Heart Leprosy Centre, Kumbakonam,India) served as healthy controls. The pa-tients included in the study were also drawnfrom both institutions, and only adults inthe age group 18-60 were included in thestudy. The socioeconomic and nutritionalstatus of the patients was not very differentfrom that of the control group.

Each of the 16 cases with ENL reactionwas given zinc sulfate (ZnSO 47H2O) orallyat a daily dose of 225 mg for a period of 6weeks. They were also given steroids as rou-tine treatment for reaction.

Sample collections. Fasting blood sam-ples were collected from the patients andhealthy controls by venipuncture, using zinc-free syringes and stainless-steel needles, andthe serum was then separated. Hemolyzedsera and hepatitis cases were not includedin the study, and extreme care was taken toavoid zinc contamination.

Methods of estimation. Serum zinc andcopper levels were estimated by atomic ab-sorption spectrophotometry using an air-acetylene flame. A Varian Techtron model-AA-1475 (Varian Instrument Division, PaloAlto, California, U.S.A.) atomic absorption

20

59, 1^ George, et al.: Scrum Zinc/Copper^ 21

THE TABLE. Scrum zinc and serum copper levels and serum zinc/copper ratios in healthycontrols and leprosy patients (mean ± S.E.).a

Group No.cases

Serum zinc level(pg/100 nil)

Serum copper level Zinc/Copper ratio(pg/l00 nil)

Healthy controls 86 113.84 ± 2.44 107.02 ± 2.42 1.101 ± 0.030Borderline tuberculoid (BT) leprosy 45 102.55 ± 3.18 118.67 ± 3.08 0.885 ± 0.033Borderline lepromatous (BL) leprosy 31 85.20 ± 3.18 130.80 ± 3.62 0.660 ± 0.025Lepromatous (LL) leprosy 117 76.92 ± 1.49 139.37 ± 3.06 0.578 ± 0.015LL cases with ENL reaction 16 68.80 ± 4.72 145.18 ± 7.19 0.485 ± 0.039LL cases with ENL reaction on oral

zinc therapy 16 126.32 ± 6.98 147.33 ± 6.51 0.912 ± 0.083

Differences in serum zinc and serum copper values and zinc/copper ratios between healthy controls and thepatient groups are all statistically significant (p < 0.001 to p < 0.01).

spectrophotometer was used throughout thestudy.

Estimation of zinc. For measurement, azinc, hollow cathode lamp was used and thecurrent adjusted to 5 ma. The monochro-meter was set at 213.9 nm with a spectralband pass (slit width) of 1.0 nm. The serumsamples were diluted 1:4 with zinc-free, tri-ple-distilled water. For a standard, pure zincmetal granules (99.9%) were dissolved 1:1in hydrochloric acid and diluted to 1 literto give a concentration of 1000/pg/m1(stock). The working standards were pre-pared at concentrations of 0.2, 0.4, and 0.6pg/ml of zinc with 10% glycerol solution tomatch the viscosity of the diluted scrum.

Estimation of copper. For estimation, acopper, hollow cathode lamp was used witha lamp current of 3.5 ma. The wavelengthwas adjusted to 324.7 nm and the spectralband pass to 0.5 nm. The serum sampleswere diluted 1:1 with triple-distilled waterto prevent protein obstruction of the burnerhead and atomizer. The stock copper stan-dard (1000 pg/ml in 1% HNO,) was pro-cured from Sigma Chemical Company, St.Louis, Missouri, U.S.A. The working stan-dards were prepared at concentrations of 1,2, and 3 pg/ml of copper using 10% glycerolsolution.

The microcomputer facility available inthe instrument was used for direct concen-tration measurement of zinc and copperfrom the standard curves created from thememory.

Statistical analysis. Statistical analysiswas carried out by applying Student's t testand Pearson and Lee's correlation coeffi-cient with the help of a computer. The p

values below 1% were considered highly sig-nificant.

RESULTSA significant decrease was noticed in the

serum zinc levels over the whole spectrumof leprosy, compared to healthy controls,the maximum decrease being seen in LLcases with lepra reaction followed by activeLL, BL, and BT cases (The Table). The meanserum zinc level (126.32 ± 6.98) in caseswith ENL reaction receiving oral zinc ther-apy was significantly higher than the levelsof the other patient groups and healthy con-trols. The differences were highly significantby Student's t test (0.001 < p < 0.005).

Compared to healthy controls, the meanserum copper levels in all of the groups ofleprosy patients were higher and the differ-ences were highly significant (The Table).Maximum increases were found in caseswith ENL reaction followed by LL, BL, andBT cases. The mean copper level (147.33± 6.51) in cases with ENL reaction on oralzinc therapy was slightly higher than themean value (145.18 ± 7.19) of active LLcases with ENL reaction, but the differencewas not significant.

The zinc/copper ratios (The Table) weregreater in LL cases with ENL reaction fol-lowed by LL, BL, and BT cases. In all fourgroups, the differences were highly signifi-cant (p < 0.001) when compared to healthycontrols. The mean ratio (0.912 ± 0.083)of zinc/copper in LL cases with ENL reac-tion treated with oral zinc sulfate was closeto the mean ratio of the healthy controls(1.101 ± 0.030), which was significantly (p< 0.001) higher than reaction cases not re-

22^ International Journal of Leprosy^ 1991

120

105

r-- 0.998

77^90IlL

LLF.^75

LL with ENL

±'60

110^120^130^140^50CONC.OF COPPER [ 09/100m1)

THE FIGURE. Scattergram of mean copper and zinclevels in healthy controls (HL), BT, BL, LL cases, andLL cases with ENL reaction.

ceiving oral zinc therapy. When comparedto the LL cases, the zinc/copper ratios of allthe other types of leprosy, including ENLreaction cases on oral zinc therapy, weresignificantly different, with p values be-tween 0.1% and 5%.

The Figure shows a very good negativecorrelation (r = —0.998) between mean se-rum zinc and copper levels from healthycontrols to LL cases with ENL reaction.

DISCUSSIONThe findings of the present study, signif-

icant reduction in the serum levels of zincand simultaneous increases in the serumlevels of copper in leprosy patients acrossthe spectrum of the disease, in general con-firm the observations of earlier investiga-tors (8, 9, I I, 30s) These differences in the se-rum levels of the two minerals could reflectthe difference between the absorptive pro-cesses of the two minerals in the stomachand in the duodenum ( 29). However, itshould be mentioned that the serum copperlevels obtained in the present study areslightly lower for both the controls and thepatient groups than those reported by earlierworkers (8 ' 30). This may be explained by thegreater sensitivity of the atomic absorptionspectrophotometry used in the present studycompared to the colorimetric methods usedearlier.

It is interesting to note that in the presentstudy a very good negative correlation was

obtained between serum zinc and copperlevels in the healthy controls and in the LLcases with ENL reaction (The Figure).

Low serum zinc levels have been foundin many conditions associated with de-pressed CMI ( 6 ' 14). In leprosy, there is agradual decrease in the serum zinc level(24.30) and depression in CMI from the TTto the LL end of the leprosy spectrum ( 3 . 4).It has been suggested that the increasingbacterial load in the macrophages throughthe leprosy spectrum could produce hypo-zincemia by increasing the liberation of theleukocyte mediator (LEM)-like substancewhich stimulates zinc redistribution withhepatic uptake, thus reducing serum zincconcentration (24). Serum zinc deficiency hasbeen reported in alcoholism (26,2x) and incirrhosis of the liver ( 27). Alteration in liverfunction in leprosy could be an inducingfactor in the very low scrum zinc levels ob-served in those with the disease. Dapsonetreatment can also contribute to the reduc-tion of the serum zinc level by competingfor protein sites, as suggested by Oon, et al.(I1) .

As regards the effect of oral zinc therapyin cases with ENL reaction, the serum levelsof zinc found in the present study are higherthan those reported by Venkatesan, et al.( 31 but are comparable to those obtainedby Mathur, et al. ( 10). This could be attrib-uted to a longer oral zinc therapy (6 weeks)in the present study than in the earlier in-vestigation by Venkatesan, et al. (2 weeks).No reduction was noticed in serum copperlevels in cases with lepra reaction receivingoral zinc therapy.

It may be mentioned in passing that thepatients included in the present study mayhave different dietary patterns (regionalvariations) from those of earlier studies.However, the results indicate that these dif-ferences do not affect serum copper and zinclevels in leprosy patients.

Mathur et al. (9 ' 10) have reported bene-ficial effects, such as faster clinical improve-ment, rapid fall in the bacterial index (BI),activation of CMI, and low incidence ofENL, when oral zinc was given as an adjunctto conventional leprosy chemotherapy. Theoptimal duration of zinc therapy, along withcareful monitoring of clinical manifesta-tions, CMI-function testing, and serum ley-

59, 1^ George, et al.: Serum Zinc/Copper^ 23

els of zinc, may help in understanding thereal significance of oral zinc therapy in lep-rosy, particularly in ulcerating reactionalstates.

SUMMARYSerum zinc and copper levels and zinc/

copper ratios were studied in 86 healthycontrols, 45 cases of borderline tuberculoid(BT), 31 cases of borderline lepromatous(BL), 117 cases of lepromatous (LL) leprosypatients, 16 cases with severe erythema no-dosum leprosum (ENL) reaction, and 16cases with ENL reaction receiving oral zinctherapy. A significant reduction in serumzinc levels was noticed in all types of lep-rosy, the maximum decrease being seen incases with ENL reaction. Conversely, thecopper levels were significantly increasedfrom BT to LL cases with ENL reaction ina progressive manner. A very good negativecorrelation (r = — 0.998) was noticed be-tween mean serum zinc and copper levelsfrom healthy controls to active LL cases withENL reaction. After oral zinc therapy, theserum zinc levels were significantly in-creased in all of the 16 LL patients with ENLreaction. In contrast, the copper levels werenot decreased, indicating that oral zinc ther-apy can restore normal zinc levels in leprosypatients but is unable to reduce the in-creased copper levels.

RESUMENSe estudiaron los niveles de zinc y cobrc, y la relaciOn

zinc/cobre, en 86 controles sanos, 45 casos de lepraintermedia-tuberculoide (BT), 31 casos lepromatosossubpolares (BL), 117 casos lepromatosos (LL), 16 casoscon severa reacciOn tipo eritema nudoso leproso (ENL),y 16 casos con reacciOn ENL bajo tratamiento con zincoral. En todos los casos se observe, una significantereducciOn en los niveles sericos de zinc pero esta fuemaxima en los casos con reacciOn ENL. Por el contra-rio, los niveles de cobre aumentaron significativa- yprogresivamente de los casos BT a los casos LL conreacciOn ENL. Yendo de los controles sanos a los casosLL activos con reacciOn ENL, se note, una buena co-rrelaciOn negativa (r = —0.998) entre los niveles me-dios de zinc y cobre sericos. Despues de la terapia conzinc oral, los niveles sericos de zinc estuvieron signi-ficativamente elevados en los 16 pacientes LL con reac-ciOn ENL pero sus niveles de cobre no estuvieron dis-minuidos. Esto indica quc quc la terapia con zinc oralpuede restaurar los niveles normales de zinc en lospacientes con lepra pero es incapaz de rcducir los ni-veles de cobrc serico.

RESUME

Les taux de zinc et cuivre seriques et les ratios zinc/cuivre ont etc etudies chcz 86 temoins, 45 cas de lepreborderline tuberculoide (BT), 31 cas de lepre borderlinelepromateuse (BL), 117 patients presentant une leprelepromateuse (LL), 16 cas ayant une reaction severed'Orytheme noueux lepreux (ENL) et 16 cas avec unENL rccevant un traitement a base de zinc par voieorale. Une diminution significative des taux de zincserique fut notee pour tous les types de lepre, la diminu-tion maximale etant vue parmi les cas de reaction ENL.Au contraire, les taux de cuivre êtaient augmentes demaniere signficative et progressivement depuis les casBT jusqu'aux LL presentant une reaction ENL. Unetres bonne correlation negative (r = —0, 998) fut trou-vee entre les taux seriques moyen de zinc et de cuivredepuis les temoins en bonne sante jusqu'aux maladesLL actifs avec une reaction ENL. Apres traitement auzinc par voie orale, les taux seriques de zinc etaientsignificativement augmentes chez tous les 16 patientsLL avec une reaction ENL. Par contraste, les taux decuivre n'êtaient pas diminues, indiquant qu'un trai-tement au zinc par voie orale peut retablir des tauxnormaux de zinc chcz les malades de la lepre, mais nepeut faire diminuer les taux augmentes de cuivre.

Acknowledgments. The authors are thankful to Dr.A. V. Paul, CLT&RI, CGL, Chief Medical officer, andDr. A. Ramachandran, Asst. Director (Clinical), forarrangement of patients for the study. The technicalassistance of Mr. M. Rajandran, D. Krishnan, and K.Pandi is gratefully acknowledged. Our thanks are alsodue to Mr. S. Seshadri for his secretarial assistance.

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