approximately 2 billion people have latent infection , 8 million would develop active tuberculosis annually , and 2 - 3 million would die due to tuberculosis . with this resurgence , cases with extrapulmonary tuberculosis
approximately 10 - 11% of extrapulmonary tuberculosis involves joints and bones , which is approximately 13% of all tuberculosis cases .
collagens are major structural proteins of the extracellular matrix , joints , and bones and their correct structure is crucial for the proper functioning of locomotor apparatus .
both tuberculosis per se and its chemotherapy with antitubercular drugs could cause pronounced changes in collagen 's contents and structure [ 1 , 2 ] . investigation of these changes is very important for improving first - line antitubercular therapy and minimization of its adverse effects .
previously we have demonstrated putative changes in rat bone and skin type i collagens amino acid contents with using different doses of pyrazinamide [ 2 , 3 ] .
type ii collagen has been classically recognized as the major collagenous component of cartilage .  
the aim of present study was to investigate potential effect of pyrazinamide on male rats cartilage type ii collagen amino acid composition .
extraction of proteoglycans was carried out by 100  ml 2  m mgcl2 , 0.05  m tris ( ph 7.6 ) during 3 days .
collagen was extracted by 100  ml 0.1  m acetic acid ( ph 2.5 ) with pepsin .
pepsin ( 20  mg / g of tissue ) was added into this solution and mixtures were left for 3 days in refrigerator at 4c .
after that for pepsin inactivation ph in each mixture was neutralized by addition of powdered crystalline tris ( to ph 7.6 ) .
fractionation of pure type ii collagens was carried out by growing concentrations of nacl according to method .
protein fraction from pellet which was formed at 4.4  m nacl concentration contained type ii collagen .
fractions were separated by centrifugation ( 65000  g , 60  min , at 4c ) .
obtained pellets were recrystallized ( 3 times ) by dialysis ( against 15% kcl in 0.02  m nahpo4 at 4c ) and centrifugation ( 65000  g , 60  min , at 4c ) .
collagen fractions were hydrolyzed : 24  h , 6  n hcl , 105c .
their amino acid compositions were analyzed by ion exchange chromatography on the amino acid analyzer aaa-881 ( czech republic ) .
in statistical processing of experimental data mean of corresponding parameter ( for each animal ) was used as independent variable .
the obtained data were calculated by one - way analysis of variance ( anova ) .
  changes in male rat cartilage type ii collagen amino acid contents induced by pyrazinamide were profound as compared to control ( table 1 ) .
statistically significant changes were registered in cartilage collagen with pyrazinamide administration at dose 1000  mg / kg for 6 amino acids and at dose 2000  mg / kg for 6 amino acids .
cartilage type ii collagen of male rats with pyrazinamide at dose 1000  mg / kg contains lower contents of alanine ( 12.3% ) and isoleucine ( 37.8% ) simultaneously with higher contents of serine ( + 46.5% ) , glutamic acid ( + 31.0% ) , and leucine ( + 46.6% ) .
collagen of rats with pyrazinamide at dose 2000  mg / kg contains lower contents of arginine ( 25.9% ) and alanine ( 31.7% ) simultaneously with higher contents of serine ( + 65.6% ) , valine ( + 32.7% ) , and leucine ( + 40.6% ) . for the majority of amino acids
our experiments demonstrated presence of qualitative changes in male rats ' cartilage type ii collagens with pyrazinamide ( in comparison with norm ) ( table 1 ) . with pyrazinamide
administration possibly could be formed cartilage type ii collagen molecules with changed surface charge ( changes in number of arginine , serine , and glutamic acid residues ) , rigidity ( changes in quantity of alanine , valine , isoleucine residues ) , number of specific loci responsible for cell adhesion , interaction with chaperons , and procollagen processing to collagen ( changes in arginine residues ) [ 814 ] .
such collagen molecules changes could hence affect the properties of connective tissues , mineralisation processes , and calcium metabolism .
comparative analysis of present results with our previous data on skin and bone type i collagens demonstrated analogous character of changes in regard to contents of serine , glutamic acid , alanine , valine , and leucine residues [ 2 , 3 ] .
this could be the evidence of existence some general mechanisms of pyrazinamide effects on collagen 's contents and structure .
moreover , having compared these data with our previous experiments , we found out analogous character of these changes in regard to changes of free serine , glutamic acid , alanine , valine , and leucine contents in liver , kidney , lung and spleen pools with different doses of pyrazinamide .
thus adverse effects of pyrazinamide ( this widely used antitubercular drug ) are much more serious and more profound than it was considered earlier . among this pyrazinamide treatment could cause qualitative changes in nucleic acid molecules , change their length , and structure .
we can suppose that such changes could be caused by pyrazinamide via its influence on nucleic acids ( coding information for this proteins synthesis ) as it was mentioned previously [ 2 , 9 , 10 ] . in our previous experiments we demonstrated epigenetic changes induced by pyrazinamide treatment , pyrazinamide - mediated alterations in male rats dna fragmentation processes , bone type
i collagen amino acid composition , spermatogenesis indices , reproductive capability , and posterity antenatal and postnatal development . besides these , on changes in collagen metabolism and structure pathologic changes in amino acid metabolism could also be affected . and at last , due to
collagen genes polymorphism [ 1114 ] , collagen structures contain in norm 4 different -chains of the same type in different concentrations .
pathology   changed concentrations in which these 4 different -chains of the same type of collagen are present in connective tissue structures .
possibly pyrazinamide - caused disturbances in amino acid compositions in our experiments could be a result of such changes in transcription rates of genes coding different -chains from the same type collagen superfamily as it was previously demonstrated for other pathology .
 
 introduction . current therapeutic regimens with first - line antitubercular 
 agents are associated to a high rate of adverse effects which could cause pronounced changes in collagen 's contents and structure . 
 investigation of these changes is very important for optimization of antitubercular therapy and minimization of treatment - caused harm . 
 the aim of present paper was to investigate potential effect of pyrazinamide on male rats ' cartilage type ii collagen amino acid composition . 
 materials and methods . 
 wistar albino male rats ( 160200  g  b.w . ) were divided into three groups : i  received pyrazinamide per os at a dose of 1000  mg / kg  b.w./day ; ii  at a dose of 2000  mg / kg  b.w./day , in both groups it was given for 60 days ; iii  control . after 60 days of the experiment , 
 rats of the experimental ( groups i and ii ) and control groups were sacrificed and the amino acids contents of male rat cartilage type ii collagens were determined using amino acid analyzer . 
 results and discussion . 
 the study of pyrazinamide effects ( administered in different doses ) on rat cartilage type ii collagen amino acid contents demonstrated presence of dose - dependent pyrazinamide - mediated quantitative and qualitative changes in these rat extracellular matrix proteins in comparison with control . 
1. Introduction
2. Materials and Methods
3. Results and Discussion