Manganese poisoning: Clinical and biochemical observations

ENVIRONMENTAL

RESEARCH

Manganese

7,374-380

Poisoning:

( 1974)

Clinical

and

Biochemical

Observations

SATYA V. CHANDRA, P. K. SZTH, AND J. K. MANKESHWAR Industrial

Toxicology

Research India

Centre, Lucknow ( Ltd). Nagpur

Received

June

and Manganese

Ore

28, 1973

Clinical and biochemical studies were conducted in twelve cases of suspected manganese poisoning. On the basis of neurological observations they were divided into three grades of poisoning, mild, moderate and severe. A significant increase in serum calcium and adenosine deaminase levels was observed in cases of mild and moderate grades of poisoning, respectively. In a case of severe poisoning the increase in serum calcium was twofold and that of adenosine deaminase threefold that in normal volunteers. Serum inorganic phosphates, alkaline phosphatase and proteins were not altered. No relationship could be established between the period of exposure to manganese and severity of poisoning. The mechanism of hypercalcemia and increase in adenosine deaminase in serum has been discussed. It is suggested that serum calcium levels could be of great manganese poisoning in its early stages. Significant changes levels did not appear until moderate poisoning occurred.

significance in adenosine

in detecting deaminase

Chronic manganese toxicity manifests itself in the form of a neurological syndrome resembling Parkinsonism (Balani et al., 1967). It is a chronic disabling condition which is preceded by certain functional disturbances termed as “premanganism” ( Rodier, 1955). Despite numerous investigations, no specific tests are known for detecting casesin the early stage, when the disorder is possibly reversible. Recently we have reported that in experimental manganese toxicity, alterations in serum calcium, inorganic phosphates and alkaline phosphatase occurred much earlier than the pathological changes in the brain of rabbits treated intratracheally with managanese dioxide (Chandra, 1972; Chandra et al., 1973). We have also reported that adenosine deaminase is significantly increased in cerebrospinal fluid in experimental manganese poisoning (Mustafa and Chandra, 1972). The present study is an attempt to explore similar biochemical alterations in human manganese poisoning in order to assesstheir s;gnificance as early diagnostic measures. SUBJECTS

AND

METHODS

In this study 12 male patients of suspected manganese poisoning and 20 healthy volunteers of the same age group were investigated. All patients were underground drillers in Balaghat and Ukwa manganese mines near Nagpur. Complete case histories including family history of nervous disorders, presence of venereal diseases, tuberculosis and alcoholism were recorded. General physical and complete neurological examinations were carried out in all cases. Only two patients could write their names, therefore writing disturbances were investigated 374 Copyright All rights

@ 1974 by Academic Press, Inc. of reproduction in any form reserved.

MANGANESE

POISONING

375

by asking them to draw triangles, squares and circles. Based on neurological findings, the cases were classified into three grades of poisoning: mild, moderate, and severe. These grades corresponded to the three phases of the disease described by Rodier ( 1955). Blood and urine samples of all the subjects were collected and the investigations were carried out as described below: 1. Blood cell counts and hemoglobin estimation (Sahli’s method) for general health assessment. 2. Serum Calcium and inorganic phosphates were determined as described ( 1925), respecby Clark and Co&p ( 1925) and Fiske and SubbaRow tively. The activity of alkaline phosphatase and adenosine deaminase was measured by the method of King and Armstrong (1934) and Mustafa and Tewari ( 1970)) respectively. Total proteins were estimated according to the procedure of Lowry et al. ( 1951). 3. Manganese content in serum was determined by an atomic absorption spectrophotometer on a Varian Techtron model AA 120. Samples were prepared by the dry ashing procedure as described by Donaldson et al (1973). After ashing samples were dissolved in 1 N hydrochloric acid and read at 279.5 nm. 4. Urine was examined by routine laboratory tests. Calcium contents were measured in 100 ml of urine (before meals samples) by the method of Clark and Collip (1925). RESULTS

History and Clinical Findings No significant findings in the family history and patient’s past illness were recorded. All twelve cases of manganese poisoning were below 40 years of age, and the period of exposure to manganese varied from four to sixteen years. After grading, 3 casesshowed disorders of mild poisoning, 8 caseshad moderate grade of poisoning and one case showed the symptoms and signs of severe poisoning. A summary of significant neurological findings in various grades of poisoning is given in Table 1. Two cases, one of mild poisoning and the other of a severe grade showed emotional instability; sudden laughter or crying occurred without apparent reason. Two cases, one from the group with mild and one from the group with moderate symptoms complained of impotence. The abnormality in walking in 8 cases of moderate poisoning varied from mild rigidity to a characteristically slow, spastic staggering, and sometimes high stepping gait. Walking backwards was difficult in most of the cases. The typical “cock walk” as described by von Jaksch (von Oettingen, 1935) was seen only in the one severe case of poisoning (Fig. 1). Tremors in hands and fingers were noticed in all the cases of moderate and severe grades of poisoning. Tremors were fine, intermittent and were particularly evident on the hyperflexion of arms and hands. Diminished muscular strength, particularly in lower extremities, was also present in all these patients. The abnormality in drawing circles, triangles

376

CHANDRA,

SETH

AND

TABLE

MANKESHWAR

1

SUMMARY OF SWNIFICANT CLINICAL OBSERVATIONS VARIOUS GRADES OF MANGANESE POISONING

No. of cases

Age range (years )

Duration of exposure range beam)

3

25-38

4-10

Mild

8

21-35

5-16

Moderate

26

6

Grades of poisoning

Severe

IN

Significant disorders Asthenia Muscular pains Mental excitability Sexual symptoms Fatigue Clumsiness of movements Clumsiness of movements Speech disorders Difficulty in walking Change in facial expressions Exaggeration of reflexes in lower liibs Tremors Adiodokinesis Tremors “Cock walk” Walking backwards not possible Muscular hypertonia in extension Spasmodic laughter Masklike face

and squares was noticed in 6 casesof moderate and one case of severe poisoning (Fig. 2). Fundus examination of the eyes and sensation to pin prick showed no abnormality; there was also no muscular wasting. Laboratory Investigations 1. Blood cell counts and bmoglobin. In all twelve casesof manganesepoisoning the red cell count and hemoglobin values ranged from 3.5 to 4.8 million mm3 and 11.0 to 14.5 g/100 ml, respectively. The range of total white cell count in both groups was 7000 to 11,000 mm3 with normal percentage of white cell forms in differential counts. The range of red blood cells and hemoglobin in twenty healthy volunteers was 5.0 to 5.6 million mm3 and 15.0 to 17 g/100 ml, respectively. 2. The mean values of serum calcium, inorganic phosphates, alkaline phosphatase, adenosine deaminase and proteins in cases of manganese poisoning as compared with healthy volunteers are given in Tables 2 and 3. The increase in serum calcium is gradual, in caseshaving a mild to moderate degree of poisoning, while the increase in the severe case is twice the amount present in healthy volunteers. The serum adenosine deaminase showed significant increase in cases

MANGANESE

FIG.

1.

This

photograph

illustrates

the

377

POISONING

peculiar

pattern

of gait

“cock

walk”

in manganese

poisoning. having moderate grade of poisoning while the increase in one severe case was more than three times that of the normal levels. 3. Manganese concentration in serum ( pg/lOO ml). Manganese concentrations in serum of healthy volunteers is 3.52 k 0.10. The concentrations of manganese in three grades of poisoning are 6.85 + 1.07, 4.1 2 0.64, and 7.5, respectively.

FIG.

2.

drawings by

(a) Triangle, square and circle drawn patients of manganese poisoning.

by a healthy

person.

(b and

c)

Abnormal

378

SERUM

CHANDRA,

C.~LCKIM

AND INORQANIC (MEAN

SETH

AND

TABLE 2 PHDSPH.~TES IN PATIENTS VALUES k SE mg/lOOml)

Subjects volunteers (12)’ having mild poisoning

Patients

having

moderate

poisoning

(8)

severe

poisoning

(1)

n Figures in parenthesis represent number * Significance of difference between mean

POISONING

Inorganic phosphates

9.1 13.13 P < 14.07

(3)

P having

OF MANGANESE

Calcium

Healthy Patients

Patients

MANKESHWAR

* 0.15 * 0.63 0.01* f 0.94 < O.Olb 20.0

of cases. values of patients

Solubility product

3.59 2.96

3~ 0.56 zk 0.49 n.s. 2.72 + 0.11 ns. 2.04

33 39 38 41

and controls.

There is no correlation between the concentrations of manganese and the severity of symptoms. 4. Urine. The mean values of excretion of calcium in healthy volunteers is 3.5 mg 5 O.Ol/lOO ml. The excretion of calcium in mild and moderate grades of poisoning is 3.0 f. 0.07 mg/lOO ml while in the one severe case the excretion was 1.0 mg/lOO ml. There was no other abnormal finding in the urine in all these cases. DISCUSSION

The case histories and clinical observations leave little doubt that the twelve cases in the present study are the victims of manganese poisoning in various grades. These findings show striking similarities to the clinical observations in cases of manganese poisoning reported from India (Niyogi, 1958; Balani et al., 1967) and the other parts of the world (Rodier, 1965; Emara et al., 1971). There was no relationship with the period of exposure and severity of poisoning which is in accordance with the reports of Penvalver (1955) and Emara et al. (1971). Biochemical investigations conducted in these cases of manganese poisoning SERUM

ALKALINE PATIENTS

PHOSPHATASE, OF MANGANESE

Subjects Healthy Patients

volunteers (12ja having mild poisoning

Patients

having

moderate

Patients

having

severe

(3)

poisoning poisoning

(8) (1)

0 Figures in parenthesis represent number * Significance of difference between mean

TABLE 3 ADENOHNE POISONING

DEAMINASE AND PROTEINS (MEAN VALUES + SE!

Alkaline phosphatase K.A. units

Adenosine deaminase units/100 ml

9.75 12.6

8.56 8.6

+ 0.72 C 1.4 n.s. 11.4 + 1.1 ns. 8.0

of cases. values of patients

+ 0.60 + 0.22 n.s. 12.7 + 0.60 P < 0.016 28.0

and

controls.

IN

Proteins g/100 ml 8.7 7.99

f 0.14 f 0.20 ns. 6.80 f 0.23 n.8. 8.0

MANGANESE

POISONING

379

show a significant increase in the concentration of serum calcium in mild and moderate poisoning and a very high value (20.0 mg/lOO ml) in one severe case. There appears to be gradual increase in the level of serum calcium as the disease progresses. The steady increase may perhaps be the reason for the absence of symptoms of hypercalcemia even if a very high value of serum calcium is present, as in the case of severe poisoning. The gradual increase in serum calcium in clinical cases is in accordance with the observations in experimental manganese toxicity in rabbits, where steady increase occurred from two to eight months after intratracheal administration of manganese dioxide (Chandra et cd., 1973). Furthermore, the increase in the level of serum calcium is independent of the level of inorganic phosphates and proteins which remains within normal limits. The excretion of calcium is normal in all the cases except the slightly lower excretion in the severe case. The significance of this singular finding can not be ascertained and requires further studies in similar cases of manganese toxicity. The mechanism of this dissociation between hypercalcemia and calcium excretion in manganese toxicity is not understood. Further studies are needed to understand the role of the parathyroid gland in altering the serum calcium level, especially when the excretion of this element remains normal and no changes have been seen in inorganic phosphates concentrations. The increase in the level of adenosine deaminase in cerebrospina1 fluid of rabbits treated intratracheally with manganese dioxide has been reported earlier, though its significance could not be ascertained (Mustafa and Chandra, 1972). In present studies there is also a significant increase in the activity of this enzyme in serum in the cases of moderate grades of poisoning, while there is a threefold increase in the severe case as compared with the activity in normal subjects. The clinical significance of adenosine deaminase levels is not known. It may be possible that in early stages of manganese poisoning this enzyme leaks out from the nervous tissue into the cerebrospinal fluid and serum due to damage of neurones at the biochemical level. Normal or only slightly higher value of manganese in serum were observed in our cases. It has been observed that even relatively large ingested amounts, 800 mg for three months do not alter the serum manganese level (Mahoney et al., 1969). It is also known that about 70% of the blood manganese leaves the circulation every minute (Cotzias et al., 1966). Normal manganese levels in blood have been reported in manganese poisoning cases by other workers (Emara et al., 1971; Bellare, 1967). This observation may suggest that estimation of manganese level in serum has no significance in detecting manganese poisoning. Our results suggest that determination of serum calcium levels could be of great significance in detecting manganese poisoning in early stages. In cases with moderate degrees of poisoning the adenosine deaminase levels are also significantly increased. However, further studies on a greater number of cases are needed to determine the mechanism of these biochemical alterations, and to define their correlation with the toxic action of manganese.

380

CHANDRA,

SETH

AND

MANKESHWAR

ACKNOWLEDGMENTS The authors are grateful to Dr. S. H. Zaidi, Director, Industrial Toxicology Research Centre, and Dr. G. J. Mohan Rao, Scientist-in-Charge, Central Public Health Engineering Research Institute, for their keen interest and valuable suggestions in this work. Field and laboratory facilities provided by Mr. P. K. Yennawar, Head of Air Pollution Division CPHERI, Nagpur and facilities for manganese determination provided by Dr. C. P. Sharma, Department of Botany, Lucknow University, are gratefully acknowledged. We thank the laboratory staff of MOIL, Nagpur, photography section, CPHERI, Nagpur and to Mr. R. S. Srivastava for technical assistance. REFERENCES S. G., UMARJI, G. M., BELLARE, R. A., AND MERCHANT, H. C. (1967). Chronic manganese poisoning. J. Postgraduate Med. (Bombay) 13, 116-122. BELLARE, R. A. ( 1967). Studies in manganese poisoning. Doctoral thesis, Bombay University. CIIANDRA, S. V. ( 1972). Histological and histochemical changes in experimental manganese encephalopathy in rabbits. Arch. Toxlkol. 29, 29-38. CHANDRA, S. V., IMAM, Z., AND NAGAR, N. ( 1973). Significance of serum calcium, inorganic phosphates and alkaline phosphatase in experimental manganese toxicity. Ind. Health, in press. CLARK AND COLLIP ( 1925). “Hawk’s Physiological Chemistry” (B. L. Oser, Ed. ), 14th ed. McGraw-Hill, London, p. 1133. CLARK AND COLLIP ( 1925 ) . “Hawk’s Physiological Chemistry” (B. L. Oser, Ed.), 14th ed. McGraw-Hill, London, p. 1263. COTZIAS, G. C., MILLER, S. T., AND EDWARDS, J. ( 1966). Neutron activation analysis: The stability of manganese concentrations in human blood and serum. J. Lab. Clin. Med. 67, 836-841. DONALDSON, J., PIERRE, T. ST., MXNNICH, J. L., AND BARBEAU, A. ( 1973). Determination of Na’, K’, Mg*+, Cu’+, Zn’+ and Mn*+ in rat brain regions. Canad. J. Biochem. 51, 87-92. EMARA, A. M., EL-GHAWABI, S. H., MADKOUR, 0. I., AND EL-SAMRA, G. H. ( 1971). Chronic manganese poisoning in the dry battery industry. Brit. J. Ind. Med. 28, 78-82. FISKE, C. M., AND SUBBAROW, V. ( 1925). “Hawk’s Physiological Chemistry” (B. L. Oser, Ed.), 14th ed. McGraw-Hill, London, p. 1113. KING, E. J., AND ARMSTRONG, A. R. ( 1934). “Hawk’s Physiological Chemistry” ( B. L. Oser, Ed. ) , 14th ed. McGraw-Hill, London, p. 1119. LOWRY, 0. H., ROSELROUGH, N. J., FARR, A. L., AND RANDALL, R. J. ( 1951). Protein measurement with Folin Phenol reagent. J. Biol. Chem. 193, 265-275. MAHONEY, J. P., SARGENT, K., GRELAND, M., AND SMALL, W. ( 1969). Studies on manganese. Determination in serum by atomic absorption spectrophotometry. Clin. Chem. 15, 312-322. MUSTAFA, S. J., AND TEWARI, C. P. ( 1970). Latent adenosine deaminase in mouse brain. Biochem. Biophys. Acta. ( Amst ) 198, 93-100. MUSTAFA, S. J,, AND CHANDRA, S. V. ( 1972). Adenosine deaminase and protein pattern in serum and cerebrospinal fluid in experimental manganese encephalopathy. Arch. Torikol. 28, 279-285. NIYOGI, T. P. ( 1958). Chronic manganese poisoning. Indian J. Ind. Med. 3, 3-13. PENALVER, R. ( 1955). Manganese poisoning. The 1954 Ramazzini Oration. Ind. Med. Surg. 24, l-7. RODIER, J. ( 1955). Manganese poisoning in Moroccan Miners. Brit. J. Ind. Med. 12, 21-35. VON OETTINGEN, W. T. ( 1935). Manganese: It’s distribution, pharmacology and hea!th hazards. Physiol. Rev. 15, 175-201. BALANI,