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Biochemical studies on pulmonary candidiasis in guinea pigs
Toudcan, Vd 19, Na 4, pp . 370 372, 1981 . Rin~d in En8laod .
0041-0101/BI~WOS70-03 502.00/0 m 1961 P~erpmon Pree Ltd.
BIOCHEMICAL STUDIES ON PULMONARY CANDIDIASIS IN GUINEA PIGS AML K, JAISWAL Division of Biochemistry and Food Science, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India (Accepted jor publication 16 March 1981) A. K. JAISWAL. Biochemical studies on pulmonary candidiasis in guinea pigs. Toxicon 19, 570-572, 1981 .-Biochemical changes were followed in guinea pig lungs at 30, 60 and 90 days after an intratracheal injection of 1 .0 mg of Candida aibicans. The collagen and mucopolyaaocharide contents
were significantly increased 90 days after infection, while the suocinate dehydrogenase activity was significantly decreasod at 90 days. The lactate dehydrogenase activity and lactic acid content showed a continuous increase following infection while the pyruvic acid levels were lowered.
EXPF1tIb1ENrAL pulmonary candidiasis has been reported in rabbits (KUROTCHKIN and LIM, 1963 ; Evnxs and W1rnvER,1954 ; F1:L1sAT1 et a1.,1959) and guinea pigs (URSO and CAPOCACCIA, 1952 ; VoGeL and KRexL,1957). Detailed pathological studies by ZAIDI et al . (1973) revealed
widespread congestion, edema and acute inflammatory reaction in lungs of rhesus monkeys infected with Candida albica~ts. However, the biochemical basis for these effects has not been studied. We now report on biochemical studies in guinea pig lungs after intratracheal inoculation with Candida albicans . Canditia albicans was obtained from Antibiotics Division of Central Drug Research Institute, Lucknow. The suspension for inoculating the guinea pigs was prepared from a 4872 hr old culture grown on Sabouraud's agar medium A weighed amount of organism, scraped from the surface of the plates was then shaken for 5 min with 0.85 % sterile normal saline in a bottle containing glass beads. Suitable dilutions of the suspension were then made in sterile normal saline. Thirty male guinea pigs (300-350 g) obtained from the Small Laboratory Animal Division of Industrial Toxicology Research Center at Lucknow were used. Füteen guinea pigs were intratracheally inoculated with 1.0 mg of Candida albicans in 1.5 ml of sterile normal saline and the remaining 15 guinea pigs received 1.5 ml of saline. The experimental as well as control animals were kept on Hindustan Lever guinea pig pellet diet supplemented with green vegetables ad libitum. Five animals from each group were sacrificed by exsanguination at 30, tí0 and 90 days after infection ; their hmgs taken out and washed in normal saline. A weighed portion of the fresh lung was dried at 110°C to constant weight and 25 mg aliquots were used for collagen estimation according to the method Of S~rEGEMAxIV (1958) as described by ZAII)1 and KAw (1970). Mttcopolysaccharides were estimated as glycosamine 570
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according to the method of ASIiwELL (1957). Phospholipid content was estimated by the method of Wooz-roN (1964) . Another portion of the lung was cut into small pieces and homogenized in distilled water under chilled conditions using a Potter Elvehjem homogenizer. After straining through muslin cloth (mesh < 150), the biochemical estimations were made on the whole homogenate . Suocinate dehydrogenase and lactate dehydrogenase activities were estimated according to the procedures of SLATER and BONNER (1952) and KORNHERG (1955) respectively. Lactic acid and pyruvic acid contents were estimated by the lY]ethOd Of BARKER and SUMMERSON (1941) and FRIEDEMANN (1957). The fresh weight and protein content ofthe lung tissue did not show any significant change due to Candida albicans infection : range of weight ; fresh weight of lung 26-3.2 g, lung protein 100-116 mg/g fresh weight of lung . Table 1 reports on the composition of guinea pig lungs ofcontrol and infected animals. The collagen and mucopolysaccharide contents were not significantly changed up to 60 days. At 90 days the collagen content increased by 25~ (P < 0.01). Similarly at this stage the mucopolysaccharide level was also increased in infected animals by 30% (P < 0.01). The phospholipid was unaffected at 30 and 60 days but there was a 30% decrease (P < 0.01) 90 days after infection. The lactic acid content was increased throughout the disease, the values in experimental animals being 30, 33 and 37% (all P < 0.01) higher than in respective controls at 30, 60 and 90 days after infection. The pyruvic acid content showed 48 and 28 decrease (P < 0.01) over the respective controls at 30 and 60 days after inoculation. At 90 days after infection the succinate dehydrogenase showed a 23~ (P < 0.01) decrease (Table 2). The lactate dehydrogenase activity increased (P < 0.01) at 90 days following infection. The increase in collagen and mucopolysaccharide content at later stages of the disease accounts for the fibrosis ofmonkey lung tissue due to Candida albicans infection (ZAII)1 et al., 1973). We also found significant changes in energy metabolism due to Candida albicans. Following infection by Candida albicans, the macrophages become active. The engulûnent and destruction ofphagocytosed particles in the macrophages needs energy provided either by oxidative phosphorylation or glycolysis . Along with this there is synthesis of collagen TABLE l . COLLAGEN, MUCOPOLYSACCHARiDE, PHOSPHOLIPID, LAC17C AND PYRUVIC ACID CONTENTS OF NORMAL AND Candtda albiCanS INFECTED GUINEA PIG LUNGS
Days after inoculation of Candide albtcans Group Collagen
Control Experimental
Mucopolysaccharide
30
60
90
11 .7 t 0 .1 12 .5 t 1 .4
13.4 f 0 .3 16.9 t 2 .4'
Control Experimental
11 .9 t 0.1 12 .0 t 2.0 0 .72 t 0.09 0.75 t 0.06
0.69 t 0.08 0.74 t 0 .03
0.70 t 0.10 0.93 f 0 .04'
Phoapholipid
Control Experimental
21 .7 t 1 .4 22 .3 f 1 .0
21 .4 t 1.0 24 .7 t 1 .4
20.4 t 1 .4 12.7 f 3.T
Lactic acid
Control Experimental
1 .17 f 0.07 1 .52 f 0.003'
1 .20 t 0.07 1 .54 t 0.11'
1 .10 f 0.08 1 .51 f 0.09'
Pyruvic acid
Control Experimental
0 .034 t 0.005 0.013 t 0.000"
All values are mean t S.E. (N = 5). Values expressed as mg/g fresh weight " P < 0.01 a, compered to corresponding control value.
0.033 t 0.006 0.027 t 0.002'
0.036 t 0 .009 0.034 t 0 .001
572
Short Communications
TABLE 2. SUCCINATE AND LACTATE DEHYDROriENA3E ACTIVITIES IN NORMAL AND Candide alólCanJ INFECTED GUINEA ÍßG LUNGS Days aller inoculation of Candida alblcans Group
30
60
90
Succinate dehydrogenase (n molen of K3Fe(CN)6 reduced/min/mg protein at 37°C)
Control Experimental
14 .7 t 1.6 14 .4 t 1.2
15.4 t 2.1 14.4 t 1.3
13.1 f 1.9 10.1 t 0.9'
Lactate dehydrogenase (nmoks of NADH oxidized/min/mg protein at 37°C)
Control Experimental
5.3 t 0.2 6.0 t 0.2
5.5 t 0.5 6.6 f 0.4
5.7 t 0.6 7.5 t 0.T
All values are mean t S.E. (N = 5). 'P < 0.01 as compared to corresponding control value.
which also requires energy. To meet the increased demand for energy anaerobic respiration increased, as shown by an increase in lactate ~hydrogenase along with lactic acid and a decrease in pyruvic acid content of the lung. The small decrease in succinate dehydrogenase activity at 90 days may be due to damage to mitochondria . The above changes may also be due to a metabolite produced by Candida albicans . Acknowledpemerus-Thanks are due to DR . P. N. VISWANATHAN, Scientist, Industrial Toxicology Research Center, Lucknow for his help and supply of some experimental material
REFERENCES
ASHWELI, G. (1957) Colorimetrie analysis ofsugars.In :Methodsin Enzymolopy, Vol. III, p. 73 (COLOWICK, S. P. and KAPLAN, N. O., Eds} New York : Academic Press. BARKER, S. B.and SUMMERSON, W. H. (1941) The colorimetric determination of lactic acid in biological material J. 6io1. Chem. 13g, 535. EVANS, W. E. D. and WINNER, H. I. (1954) The hiatogenesis of the lesions in experimental monitiasis in rabbits J. path. Badertol. 67, 531. FELTSATI, D, BASITANINI, L. and DE MTrRI, T. (1959) Antibiotics and Candlda alólcanc given by endobronchiel route. Antibiot . Chemother. 9, 744. FRiEDEMANN, T. E. (1957) Determination of ketoacids In : Methodsin Enzymology, VolIII, p. 414 (COLOWICK, S. P. and KAI'IaN, N. O, Eds~ New York : Academic Press. KoRNaeRG, A. (1955) Lactic dehydrogenaee of muscle . In : Methods to Enzymology, Vol. I, p. 441 (COLOWICK, S. P. and KAPLAN, N. O., Eda). New York : Academic Press KUROTCHKIN, T.1 . and LIM, C. E. (1963) Experimental bronchomonitiaais in sensitized rabbits. Proc. Sce. exp. Biol. 21, 332 . SLATER, E. C. and BONNER, W. B. (1952) The effect of fluoride on the succinic oxidase system. Biochem. J. 52, 185. STEGEMANN, H. (1958) Miao determination of hydroxyproline with chloramin-T and p-dimethylaminobenzaldehyde. Hoppc Seykrs Z. Physiol. Chem. 311, 41 . LIRSO, B. and CAPOCACCIA, L. (1952) Osaervazioni su un Caso di monibiasi bronchiale can guadro asmatico. Arch. Ital. Sci. Med. Trop. 31, 77 . VOGEL, R. A. and KREIIL, W. (1957) Experimental sensitization of guinea pigs with Candidaalóicans and adjuvants. Amer. Res. Tubtrc. 76, 692. WoorroN, I. D. P. (1964) Microanalysis in medical biochemistry, p. 41 . London : A. Churchill Ltd. ZAIDi, S. H. andIüw, J. L. (1970) FBect of dietarydeficiency andprotein malnutrition on the flbrogeneaia caused by silica dust in rata . Br. J. lnd. mod. 27, 250. Z.AIDT, S. H., $HANKER, R. and DOGRA, R. K. S. (1973) Experimental infectioe pneumoconiosis : Effect of asbestos dust and Candida albicans infection on the lunge of rhesus monkeys. Enotron. Res. 6, 274.
0041-0101/BI~WOS70-03 502.00/0 m 1961 P~erpmon Pree Ltd.
BIOCHEMICAL STUDIES ON PULMONARY CANDIDIASIS IN GUINEA PIGS AML K, JAISWAL Division of Biochemistry and Food Science, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India (Accepted jor publication 16 March 1981) A. K. JAISWAL. Biochemical studies on pulmonary candidiasis in guinea pigs. Toxicon 19, 570-572, 1981 .-Biochemical changes were followed in guinea pig lungs at 30, 60 and 90 days after an intratracheal injection of 1 .0 mg of Candida aibicans. The collagen and mucopolyaaocharide contents
were significantly increased 90 days after infection, while the suocinate dehydrogenase activity was significantly decreasod at 90 days. The lactate dehydrogenase activity and lactic acid content showed a continuous increase following infection while the pyruvic acid levels were lowered.
EXPF1tIb1ENrAL pulmonary candidiasis has been reported in rabbits (KUROTCHKIN and LIM, 1963 ; Evnxs and W1rnvER,1954 ; F1:L1sAT1 et a1.,1959) and guinea pigs (URSO and CAPOCACCIA, 1952 ; VoGeL and KRexL,1957). Detailed pathological studies by ZAIDI et al . (1973) revealed
widespread congestion, edema and acute inflammatory reaction in lungs of rhesus monkeys infected with Candida albica~ts. However, the biochemical basis for these effects has not been studied. We now report on biochemical studies in guinea pig lungs after intratracheal inoculation with Candida albicans . Canditia albicans was obtained from Antibiotics Division of Central Drug Research Institute, Lucknow. The suspension for inoculating the guinea pigs was prepared from a 4872 hr old culture grown on Sabouraud's agar medium A weighed amount of organism, scraped from the surface of the plates was then shaken for 5 min with 0.85 % sterile normal saline in a bottle containing glass beads. Suitable dilutions of the suspension were then made in sterile normal saline. Thirty male guinea pigs (300-350 g) obtained from the Small Laboratory Animal Division of Industrial Toxicology Research Center at Lucknow were used. Füteen guinea pigs were intratracheally inoculated with 1.0 mg of Candida albicans in 1.5 ml of sterile normal saline and the remaining 15 guinea pigs received 1.5 ml of saline. The experimental as well as control animals were kept on Hindustan Lever guinea pig pellet diet supplemented with green vegetables ad libitum. Five animals from each group were sacrificed by exsanguination at 30, tí0 and 90 days after infection ; their hmgs taken out and washed in normal saline. A weighed portion of the fresh lung was dried at 110°C to constant weight and 25 mg aliquots were used for collagen estimation according to the method Of S~rEGEMAxIV (1958) as described by ZAII)1 and KAw (1970). Mttcopolysaccharides were estimated as glycosamine 570
57 1
Short Communications
according to the method of ASIiwELL (1957). Phospholipid content was estimated by the method of Wooz-roN (1964) . Another portion of the lung was cut into small pieces and homogenized in distilled water under chilled conditions using a Potter Elvehjem homogenizer. After straining through muslin cloth (mesh < 150), the biochemical estimations were made on the whole homogenate . Suocinate dehydrogenase and lactate dehydrogenase activities were estimated according to the procedures of SLATER and BONNER (1952) and KORNHERG (1955) respectively. Lactic acid and pyruvic acid contents were estimated by the lY]ethOd Of BARKER and SUMMERSON (1941) and FRIEDEMANN (1957). The fresh weight and protein content ofthe lung tissue did not show any significant change due to Candida albicans infection : range of weight ; fresh weight of lung 26-3.2 g, lung protein 100-116 mg/g fresh weight of lung . Table 1 reports on the composition of guinea pig lungs ofcontrol and infected animals. The collagen and mucopolysaccharide contents were not significantly changed up to 60 days. At 90 days the collagen content increased by 25~ (P < 0.01). Similarly at this stage the mucopolysaccharide level was also increased in infected animals by 30% (P < 0.01). The phospholipid was unaffected at 30 and 60 days but there was a 30% decrease (P < 0.01) 90 days after infection. The lactic acid content was increased throughout the disease, the values in experimental animals being 30, 33 and 37% (all P < 0.01) higher than in respective controls at 30, 60 and 90 days after infection. The pyruvic acid content showed 48 and 28 decrease (P < 0.01) over the respective controls at 30 and 60 days after inoculation. At 90 days after infection the succinate dehydrogenase showed a 23~ (P < 0.01) decrease (Table 2). The lactate dehydrogenase activity increased (P < 0.01) at 90 days following infection. The increase in collagen and mucopolysaccharide content at later stages of the disease accounts for the fibrosis ofmonkey lung tissue due to Candida albicans infection (ZAII)1 et al., 1973). We also found significant changes in energy metabolism due to Candida albicans. Following infection by Candida albicans, the macrophages become active. The engulûnent and destruction ofphagocytosed particles in the macrophages needs energy provided either by oxidative phosphorylation or glycolysis . Along with this there is synthesis of collagen TABLE l . COLLAGEN, MUCOPOLYSACCHARiDE, PHOSPHOLIPID, LAC17C AND PYRUVIC ACID CONTENTS OF NORMAL AND Candtda albiCanS INFECTED GUINEA PIG LUNGS
Days after inoculation of Candide albtcans Group Collagen
Control Experimental
Mucopolysaccharide
30
60
90
11 .7 t 0 .1 12 .5 t 1 .4
13.4 f 0 .3 16.9 t 2 .4'
Control Experimental
11 .9 t 0.1 12 .0 t 2.0 0 .72 t 0.09 0.75 t 0.06
0.69 t 0.08 0.74 t 0 .03
0.70 t 0.10 0.93 f 0 .04'
Phoapholipid
Control Experimental
21 .7 t 1 .4 22 .3 f 1 .0
21 .4 t 1.0 24 .7 t 1 .4
20.4 t 1 .4 12.7 f 3.T
Lactic acid
Control Experimental
1 .17 f 0.07 1 .52 f 0.003'
1 .20 t 0.07 1 .54 t 0.11'
1 .10 f 0.08 1 .51 f 0.09'
Pyruvic acid
Control Experimental
0 .034 t 0.005 0.013 t 0.000"
All values are mean t S.E. (N = 5). Values expressed as mg/g fresh weight " P < 0.01 a, compered to corresponding control value.
0.033 t 0.006 0.027 t 0.002'
0.036 t 0 .009 0.034 t 0 .001
572
Short Communications
TABLE 2. SUCCINATE AND LACTATE DEHYDROriENA3E ACTIVITIES IN NORMAL AND Candide alólCanJ INFECTED GUINEA ÍßG LUNGS Days aller inoculation of Candida alblcans Group
30
60
90
Succinate dehydrogenase (n molen of K3Fe(CN)6 reduced/min/mg protein at 37°C)
Control Experimental
14 .7 t 1.6 14 .4 t 1.2
15.4 t 2.1 14.4 t 1.3
13.1 f 1.9 10.1 t 0.9'
Lactate dehydrogenase (nmoks of NADH oxidized/min/mg protein at 37°C)
Control Experimental
5.3 t 0.2 6.0 t 0.2
5.5 t 0.5 6.6 f 0.4
5.7 t 0.6 7.5 t 0.T
All values are mean t S.E. (N = 5). 'P < 0.01 as compared to corresponding control value.
which also requires energy. To meet the increased demand for energy anaerobic respiration increased, as shown by an increase in lactate ~hydrogenase along with lactic acid and a decrease in pyruvic acid content of the lung. The small decrease in succinate dehydrogenase activity at 90 days may be due to damage to mitochondria . The above changes may also be due to a metabolite produced by Candida albicans . Acknowledpemerus-Thanks are due to DR . P. N. VISWANATHAN, Scientist, Industrial Toxicology Research Center, Lucknow for his help and supply of some experimental material
REFERENCES
ASHWELI, G. (1957) Colorimetrie analysis ofsugars.In :Methodsin Enzymolopy, Vol. III, p. 73 (COLOWICK, S. P. and KAPLAN, N. O., Eds} New York : Academic Press. BARKER, S. B.and SUMMERSON, W. H. (1941) The colorimetric determination of lactic acid in biological material J. 6io1. Chem. 13g, 535. EVANS, W. E. D. and WINNER, H. I. (1954) The hiatogenesis of the lesions in experimental monitiasis in rabbits J. path. Badertol. 67, 531. FELTSATI, D, BASITANINI, L. and DE MTrRI, T. (1959) Antibiotics and Candlda alólcanc given by endobronchiel route. Antibiot . Chemother. 9, 744. FRiEDEMANN, T. E. (1957) Determination of ketoacids In : Methodsin Enzymology, VolIII, p. 414 (COLOWICK, S. P. and KAI'IaN, N. O, Eds~ New York : Academic Press. KoRNaeRG, A. (1955) Lactic dehydrogenaee of muscle . In : Methods to Enzymology, Vol. I, p. 441 (COLOWICK, S. P. and KAPLAN, N. O., Eda). New York : Academic Press KUROTCHKIN, T.1 . and LIM, C. E. (1963) Experimental bronchomonitiaais in sensitized rabbits. Proc. Sce. exp. Biol. 21, 332 . SLATER, E. C. and BONNER, W. B. (1952) The effect of fluoride on the succinic oxidase system. Biochem. J. 52, 185. STEGEMANN, H. (1958) Miao determination of hydroxyproline with chloramin-T and p-dimethylaminobenzaldehyde. Hoppc Seykrs Z. Physiol. Chem. 311, 41 . LIRSO, B. and CAPOCACCIA, L. (1952) Osaervazioni su un Caso di monibiasi bronchiale can guadro asmatico. Arch. Ital. Sci. Med. Trop. 31, 77 . VOGEL, R. A. and KREIIL, W. (1957) Experimental sensitization of guinea pigs with Candidaalóicans and adjuvants. Amer. Res. Tubtrc. 76, 692. WoorroN, I. D. P. (1964) Microanalysis in medical biochemistry, p. 41 . London : A. Churchill Ltd. ZAIDi, S. H. andIüw, J. L. (1970) FBect of dietarydeficiency andprotein malnutrition on the flbrogeneaia caused by silica dust in rata . Br. J. lnd. mod. 27, 250. Z.AIDT, S. H., $HANKER, R. and DOGRA, R. K. S. (1973) Experimental infectioe pneumoconiosis : Effect of asbestos dust and Candida albicans infection on the lunge of rhesus monkeys. Enotron. Res. 6, 274.