[FETAL LOSSES DURING THE MATURATION OF THE PLACENTA AND THE RELATIONSHIP WITH SOME PROCOAGULANT CONDITIONS.]

Among the major causes and risk factors for fetal loss are chromosomal abnormalities, genetic syndromes, placental abnormalities, thrombophilia (FVL, Fil G20210A, C677T MTHFR, PAl-1 4G /-5G), infection and inflammation (IL-3, IL-4, IL-17, IL-10), antiphospholipid syndrome, maternal diseases such as hypertension, diabetes and obesity. Pregnancy is a prothrombotic state as a result of specific physiological changes with multifactorial ethio-pathogenesis, leading to increased procoagulant factors and structural changes turned a sTasis, inflammatory component and contribution of individual genetic and acquired thrombophilic risk factors. Understanding of the molecular mechanisms of control over the process of embryogenesis, placentation and fetal development and impact of the factors of hemodtasis, inflammation and apoptosis, contributes to the application- of appropriate therapy and increase the chance of successful completion of pregnancy.

[SEX HORMONE INFLUENCE ON PERIPHERAL NATURAL KILLER CELLS COUNT].

Proper evaluation of immunological factors connected with pregnancy establishment increased the possibility for exact treatment in high risk gestation cases. Hormonal changes during an ovarian cycle may affect immune response, which is crucial for the embryonic implantation. Peripheral Natural killer (pNK) cells are key components of immune systems and their activities could be regulated by sex hormones. In the present study we investigated the effects of estrogen fluctuation on the number of NK cells in vivo during the early follicular and middle luteal phase of menstrual cycle. In 63 healthy women with at least one full term pregnancy and regular menstrual cycle with duration between 24 and 32 days, blood samples have been collected twice for investigation of CD3/CD16/CD56 positive lymphocytes. The mean pNK count in follicular phase was 11.6% with 4.7% variation. The median was 10.6%. The mean pNK count in luteal phase was 12.1% with 5.1% variation, respectively median for cell number 11.8%. The two-tailed t-test comparison did not find any statistical difference despite the slight elevation of pNK cells count in luteal phase. The insignificant variation in pNK cells count objected the suggestion to evaluate immunological status in women with adverse pregnancy outcome in specific phase of menstrual cycle.

[Implication of PAI-1 4G/5G polymorphism in recurrent implantation failure after IVF].

During implantation, an accurate balance of coagulation, fibrin deposition and fibrinolysis is mandatory for trophoblastic invasion. Inhibition of fibrinolysis after increased activity of plasminogen activator inhibitors such as PAI-1 could impair properdeep trophoblastic invasion. This study investigated correlation between increased PAI-1 levels due to gene polymorphism (PL) 4G/5G and recurrent implantation failure after IVF procedure. Sixty one women with two or more unsuccessful IVF procedure after good quality embryo transfer and 97 health women with at least one normal delivery were investigated for carrier status for PL 4G/5G (genotype 4G/4G) and serum levels of anti-cardiolipin (ACA) and anti-beta2-glycoprotein antibody level (IgG and IgM type). The prevalence of genotype 4G/4G in women with RIF was about two times higher compared to controls although the difference did not rich significance (respectively 41% and 26.8%, OR 1.9, 95%CI 0.91-3.96, p=0.09). The prevalence of polymorphism was similar after exclusion of for women with elevated levels of ACA (respectively 42.1% and 26.8%, OR 1.99, 95%CI 0.94-4.21, p=0.075). PL 4G/5G could be possible risk factor forimpaired embryo implantation. The causative hypofibrinolysis due to increased PAI-1 levels should be interpreted in context of multifactor complexity of recurrent implantation failure development. A discussion remains for fraction heparin application and endometrial receptivity modulation in very early pregnancy wastage.

[Preplacentation pregnancy loss in cases of angiotensin-converting enzyme insertion/deletion polymorphism].

The balance between coagulation and fibrinolysis processes is critical for establishment and development of early pregnancy. Angiotensin-converting enzyme (ACE) is related with plasminogen activator inhibitor-1 activity which is a key regulator in embryo implantation. Therefor polymorphisms in ACE gene and variation in ACE activity could be associated with an early pregnancy wastage risk. This study investigated carrier status for insertion/deletion (I/D) polymorphism in introne 16 of ACE gene in 71 women with two or more pregnancy loss in preplacentation period (between 10 and 14 weeks of gestation) and 75 women without pregnancy complications. DD genotype for I/D polymorphism was found respectively in 31% and 24% in patients and controls. Heterozygosity of D allele was found correspondingly in 47.9% and 54.7%. The dominant genetic model was used for allele prevalence comparison. D allele in DD genotype was not significantly prevalent in women with early pregnancy wastage compared with the control subjects, OR = 1.42, 95% CI (0.64-3.15). The study found a weak association between I/D polymorphism and preplacentation pregnancy loss. The additive effect over the pregnancy loss risk of I/D polymorphism could be supposed in a presence of other inherited or acquired factors connected with endometrial receptivity and implantation process.

[A weak association of 677 C>T polymorphism in MTHFR with recurrent embryonic loss].

Early (embryonic) pregnancy loss before 10 week of gestation (wg) could also be related with endometrial receptivity as well as with gene expression regulation in developed embryo. Methylation of genome is a key process in the gene expression. Because the methylenetetrahydrofolate reductase (MTHFR) have had significant role in methionine metabolism polymorphisms into the gene could be related with early embryonic development. This study evaluated relationship between T allele in 677 C>T polymorphism in MTHFR and recurrent embryonic loss development. One hundred six women with tree or more pregnancy loss before 10 wg and 165 women without reproductive failure have been evaluated for 677 C>T carrier status. Sixteen (15.1%) of women with pregnancy loss have had TT genotype and 54 (50.9%) are heterozygous carriers for T allele. T allele frequency was higher but not significant differ from carrier status in control group (13.9% for TT genotype and 43.9% for CT OR and 95% CI respectively 1.1, 0.52-2.3 u 1.34, 0.8-2.26, p > 0.05). T allele (in homozygous and heterozygous carriers) was in higher but not significant prevalence in patients compared with controls (66% and 57.6% respectively, OR 1.43, 95% CI 0.84-2.46, p > 0.05), This study found a weak association between T allele carrier status (both in homozygous and heterozygous state) and recurrent embryonic loss development. T allele in 677 C>T polymorphism could be considered like an agent for early pregnancy wastage only in a constellation with other risk factors influencing embryonic development.

[Reproductive problems in women with PCOS, the impact of PAI-1 carriers of 4G PAI-1 polymorphism and BMI].

Approximately 7-12% of women in reproductive age are affected by PCOS[2] and 40 to 70 percent of them are overweight contributing to the clinical picture of PCOS and increased reproductive and metabolic disorder. In order to investigate the role of PAl-1 as a possible risk factor for the development of PCOS a group of 67 women with polycystic ovarian disease and 70 healthy controls were investigated for levels of PAl-1 and carriage of the promoter polymorphism 675 4G/5G in gene of PAI-1. The correlation with BMI was checked. The results of the DNA analysis showed a high carriage of polymorphism 675 4G/4G in promoter of PAl-1 gene in women with PCOS but not significant (OR = 1.655; p = 0.141), as well in the total group of the patient (OR =1.474; p>0.05). Serum levels of PAI-1 were significantly higher in total group of patients compared to controls. The levels of PAI-1 is correlated with carriage of 675 4G/5G polymorphism in the gene for PAI-1 (r=0.534; p=0.03) as well as with BMI, like correlation coefficients were higherin the group with PCOS (0.572; p=0.04). Data from the disease history showed a higher percentage of women with reproductive problems: early pregnancy loss 48.5% and infertility 23.2%, significantly higher in the group with PCOS (58.1% compared to 32.4%). The carriers of polymorphism 4G are at greater risk for early pregnancy loss than those with 5G (61.45% as compared to 36.8%), which confirms that carriage of the polymorphism 4G/5G 675 gene PAl-1 has a specific in multifactorial pathogenesis and expression of PCOS.

[Reproductive problems in women with PCOS, the impact of PAL-1 CARRIERS OF 4G PAI -1 polymorphism and BMI].

Approximately 7-12% of women in reproductive age are affected by PCOS[2] and 40 to 70 percent of them are overweight contributing to the clinical picture of PCOS and increased reproductive and metabolic disorder. In order to investigate the role of PAl-1 as a possible risk factor for the development of PCOS a group of 67 women with polycystic ovarian disease and 70 healthy controls were investigated for levels of PAI-1 and carriage of the promoter polymorphism 675 4G/5G in gene of PAl-1. The results of the DNA analysis showed a high carriage of polymorphism 675 4G/4G in promoter of PAI-1 gene in women with PCOS but not as significant (OR = 1.6645, p = 0.141). Serum levels of PAI-1 were significantly higher in total group of patients compared to controls. The levels of PAI-1 is correlated with carriage of 675 4G/5G polymorphism in the gene for PAI-1 (R = 0.534, p = 0.03) as well as wih BMI, like correlation coefficients were higher in the group with PCOS (0.572, p = 0.04). Data from the disease history showed a higher percentage of women with reproductive problems: 61.5% (early pregnancy loss and infertility) significantly higher in the group with PCOS (70.1% compared to 54.1%). The carriers of polymorphism 4G are at greater risk for early pregnancy loss than those with 5G (61.45% as compared to 36.8%), which confirms that carriage of the polymorphism 4G/5G 675 gene PAI-1 has a specific in multifactorial pathogenesis and expression of PCOS.

[Factor V Leiden in women with repeated IVF failures].

A plenty of factors have been connected with embryo implantation and further fetus development. Recurrent implantation failure (RIF) after assisted reproductive technology (ART) forces seeking the causes of decreased endometrial receptivity. A non-haemostatic function of thrombophilic mutations such as Factor V Leiden (FVL) was considered a factor related with endometrial receptivity. One hundred eighty eight women with two or more RIF after in vitro fertilization procedures investigated for carrier status for FVL was compared with carrier status of 97 women without reproductive failure who give a birth of at least one health child. There was no significant difference in carrier status for FVL in patients and controls (5.9% and 7.2% respectively, OR 0.80, 95% CI (0.26-2.73, p>0.05). Negligible higher prevalence of FVL was fond in health subjects compared with women with RIF A slightly positive relationship was found between FVL and embryo implantation. A preliminary determination of thrombophilic status in RIF women could specify needing or rejection of anticoagulant therapy during implantation period.

[Inherited thrombophilic factors in women with secondary infertility].

Because of the presence of additional confounding factors, such as cervical incompetence or uterine infections, the impact of inherited thrombophilia in women with second infertility has been hard to assess. The evaluation of the significance of the most common inherited thrombophilic factors - Factor V Leiden (FVL), prothrombin gene mutation 20210 G > A (FII), polymorphism (PL) 677 C > T in MTHFR, PL A1/A2 in platelet glycoprotein IIb/IIIa and PAL-1 PL 4G/5G in 35 women with two or more secondary (who have given birth to at least one child) recurrent pregnancy loss (RPL) before 14 weeks of gestation compared to 70 healthy women with no history of RPL and at least one uncomplicated full-term pregnancy, has been performed. Eight out of 35 women with secondary RPL (25.7%) and 6 out of 70 controls (8.6%) have had FVL or FII 20210 G > A (OR: 3.7, 95% CI: 1.05-13.2, p = 0.038). Five (14.3%) women with secondary infertility were carriers for FVL and four (11.4%) for FII 20210 G > A, corresponding to four (5.7%) and two (2.9%) of the women in the control group. The carrier status for MTHFR 677 C > T (TT genotype), PL A1/A2 and PL 4G/5G (4G/4G genotype) was as follows: 11.4%, 28% and 30.8% in patients and 14.3%, 17.1% and 24.3% in controls without significant difference between the groups. Despite of the presence of background factors, an appreciable role of inherited thrombophilia in secondary RPL was established, which enforces thrombophilia testing and management of women with second infertility as well as women with primary RPL.

[Platelet intregrin beta3 A1/A2 polymorphism in women with stillbirth].

Maternal thrombophilia was recently discussed as possible cause for pregnancy complication, although the roles of some coagulation factors have not been clarified. Carrier status for platelet integrin beta3 polymorphism A1/A2 (PL A1/A2) was considered as possible risk factor for pregnancy complication. Seventy women with one or more stillbirth (intrauterine fetal death after 20 week of gestation) and 100 healthy control subjects were evaluated for PL A1/A2 to assess the impact of polymorphism for late pregnancy loss. The prevalence for PL A1/A2 in women with stillbirth was higher but not significantly differs from carrier status in control subjects (respectively 28.3% and 17%, OR = 1.93; 95% CI: 0.84 - 4.45). After adjustment for carrier status for Factor V Leiden (FVL) and Prothrombin (FII) gene mutation 20210 G > A the prevalence of PL 1/A2 remains a similar (28.2% O R = 1.92; 95% Il: 0.78 - 4.75). Combined carriers status for PL A1/ A2 with FVL or III 20210 G > A have had significantly higher prevalence in investigated group comparing with control subjects (respectively 20% and 2%, p < 0.0001). An independent impact of PL A1/A2 on risk of stillbirth development is not be yet established but additive role of the polymorphism in combination with other thrombophilic factors should be considered.

[Combined thrombophilic factors among women with late recurrent spontaneous abortions].

The aim of this study was to assess the role of combined thrombophilic factors carrier status for development of late recurrent pregnancy loss (RPL). The polymorphism 4G/5G (PL 4G/5G) - genotype 4G/4G in plasminogen activator inhibitor type 1 (PAI-1), Factor V Leiden (FVL) and prothrombin (FII) gene mutation 20210 G>A in 52 women with recurrent pregnancy loss between 10 and 20 weeks of gestation and in 125 healthy women with at least one uncomplicated full-term pregnancy was investigated. Combined carrier status for thrombophilic factors was more pronounce among women with RPL (7.7%) compared to control subjects (3.2%), (OR=2.52, 95% CI (0.5- 12.62), p-ns). The most common association was between FVL and PL 4G/5G (5.8% compared to 0.8% in patients and controls, OR=7.59, 95% CI (0.68 - 191.04), p-ns). Because of relatively small size of the study, the difference in carrier status between women with RPL and control subjects did not rich statistical significance. A weak association between double carrier status for inherited thrombophilic factors and RPL was established. The strong determination in larger studies of the relation between combined inherited thrombophilic status and RPL development could better specify anticoagulant prophylaxis in further pregnancy

[Plasminogen activator inhibitor type 1 activity in women with unexplained very early recurrent pregnancy loss].

The aim of the study was to assess the independent role of polymorphism 4G/5G (PL 4G/5G)--genotype 4G/4G in plasminogen activator inhibitor type 1 (PAI-1) in the development of very early recurrent pregnancy loss (RPL)--before 10 weeks of gestation of pregnancy. The polymorphism 4G/5G as well as Factor V Leiden (FVL), prothrombin (FII) gene mutation 20210 G > A and polymorphism 677 C > T in methylentetrahydrofolat reductase (MTHFR) gene was investigated in 110 women with recurrent pregnancy loss before 10 weeks of gestation and in 97 healthy women with at least one uncomplicated full-term pregnancy. A significant prevalence of PL 4G/5G in women with RPL was found in comparison to prevalence of the polymorphism in controls (41.8% versus 26.8% respectively in patients and controls, OR: 1.96, 95% CI: 1.05 3.69, p = 0.034). The difference in prevalence of the polymorphism remains still significant after exclusion of patients and control carriers of FVL, FII 202010 G > A and 677 C > T in MTHFR (the prevalence of PL 4G/5G alone was 44.1% and 24% respectively in patients and controls, OR: 2,5, 95% CI: 1,15 5, 45, p = 0.018). The found association of PL 4G/5G in PAI-1 with early recurrent pregnancy loss encourage an extension of the list of inherited thrombophilic factors with this one. This result also could have had an implication for adjustment of further prophylactic low-molecular weight heparin implication in further pregnancy to prevent a poor foetal outcome.

[Inherited thrombophilic factors in women with unexplained intrauterine fetal deaths].

The aim of the study was to investigate a relationship between carrier status for factor V Leiden (FVL), prothrombin gene mutation 20210 G>A (PTM 20210 G>A) and development of unexplained intrauterine fetal deaths (UIFD). Thirty three women with one or more UIFD and stillbirths were investigated for carriers status for FVL and PTM 20210 G>A. Women with multiple pregnancies, congenital anomalies, intrauterine infection or chorioamnionitis were excluded from the study. Control group consisted of 79 women without reproductive failure were selected and investigated. The prevalence of FVL was significantly higher in the study group (21.1%) compared with 6.3% in the control group (OR 3.98, 95% CI 1.02- 16.14, p = 0.045). The prevalence of PTM 20210 G>A was also much higher in patients (10%) than in controls (2.5%) (OR 3.85, 95% CI 0.49-35.08, p-ns). Seven patients with UIFD and other obstetrics complications (preeclampsia, placental abruption, intrauterine growth retardation) showed high prevalence (over 40%) of FVL and PTM 20210 G>A. We found an important association between UIFD and FVL and PTM 20210 G>A, although the data on PTM 20210 G>A was non-significant because of low rate of the mutation and small group of investigated women. This data serves as a background to suggest a routine testing for inherited thrombophilia in women with UIFD aiming and individual approach of preventive use of low-molecular-weight heparin to avoid obstetric complication in future pregnancy.

[High risk of recurrent spontaneous abortion during second trimester in women carriers of polymorphism A2 in platelet glycoprotein IIb/IIIa].

The aim of this study was to evaluate the role of polymorphism A2 (PLA2) in platelet glycoprotein IIb/IIIa (GP IIb/IIIa) in the development of recurrent spontaneous abortion (RSA)--miscarriages before 20th week of gestation (wg) of pregnancy. The carriage status of PLA2 in GP IIb/IIIa, single and in combination with FVL and FII G20210A was investigated in 56 women with recurrent miscarriages before 10 g, in 38 women with RSA from 10 to 20 wg and in 98 healthy women with at least one uncomplicated full-term pregnancy. The significant prevalence of carriage of PLA2 in GP IIb/IIIa in women with RSA in first 20 wg was found with high risk or miscarriage (OR = 4.32; 95% CI: 2.10-8.97, p < 0.0001). However, after adjustment for combined carriage of other thrombophilic factors (PLA2 and FVL or PLA2 with FII G20210A) the risk was still high (OR = 2.07; 95% CI: 0.98-4.40 p = 0.058), but not significant. The similar results (OR = 2.632; 95% CI: 1.140-6.104, p = 0.021) were found for women with recurrent miscarriages in the first 10 wg. The prevalence of PLA2 adjusted for combined carriage of other thrombophilic factors was also not significant. The carriage status of PLA2 in GP IIb/IIIa in women with RSA in the period from 10 to 20 wg was significantly higher as compared to controls (OR = 8.79; 95% CI: 3.477-22.605, p < 0.0001). The prevalence, adjusted for combined carriage of other thrombophilic factors (PLA2 with FVL or PLA2 with FII G20210A) was also significantly higher (OR: 2.990; 95% CI: 1.178-7.613, p = 0.018). These results confirm the impact of PLA2 polymorphism on RSA in the period of 10 to 20 wg, and its contribution to RSA in the first 10 wg in combination with other thrombophilic mutations. The results support the suggestion of testing women with miscarriages in first 20 wg for PLA2 carriage and application of appropriate prophylactic antiplatelet drug therapy for next planned pregnancy.

[Risk of thrombophilia in carriers of thrombophilic genetic factors in unsuccessful assisted reproduction].

The aim of this study was to evaluate an association of carrier status of common inherited thrombophilic genetic mutations and implantation failure after assisted reproduction (ART): IVF and ICSI. Sixty seven women with failure of embryo implantation and ninety six controls--women without obstetric complication were investigated for carriage of factor V Leiden (FVL), G20210A prothrombin gene mutation, genetic variant C677T in methylentetrahydrofolate reductase gene (MTHFR) and polymorphism A2 in platelet glycoprotein IIb/IIIa (GIPr IIb/IIIa). A significantly higher prevalence of polymorphism A2 in GIPr IIb/IIIa was found in women with implantation failure in ART compared to controls (respectively 26.1% and 12.5%; OR: 2.571, 95% CI: 1.066-6.258, p = 0.033). A higher but not significant prevalence of G20210A prothrombin gene mutation carriage was found inpatients compared to controls (respectively 5.8% and 3.13%, OR: 1.968, 95% CI 0.356-11.539). The carriage of FVL was a little but not significantly higher in controls. The carriage of genetic variant C677T in MTHFR was the same in both groups. These data suggest that polymorphism A2 in GIPr IIb/IIIa and G20210A prothrombin gene mutation could be play a role in the etiology of IVF failures and the carriers of GIPr IIb/IIIa A1/A2 and G20210A prothrombin gene mutation are at higher risk of implantation failure and not successful ART outcome. The carriage of these two genetic defects should be investigated in women undergoing IVF and the antithrombotic or anticoagulant prophylaxis should be indicated for carriers of these two factors.

[Genetic variant C677T in the MTHFR in women with recurrent early fetal loss].

The aim of this study was to evaluate correlation of carrier status for thrombophilic gene mutation--C677T in the methylenetetrahydrofolate reductase (MTHFR) and recurrent early pregnancy loss. Recently inherited thrombophilia was discussed as a predisposed factor for early recurrent fetal loss (ERFL). We investigated carrier status for C677T genetic variant in 54 women with ERFL before 10 week of gestation and 67 women with one or more successful pregnancy. It was found significant prevalence of C677T genetic variant in MTHFR in women with ERFL compared with controls (p = 0.005). The significant high prevalence of C677T genetic variant in women with ERFL suggests that thrombophilia have an increased risk of early pregnancy loss and possibly, although the definition of the magnitude of risk will require prospective longitudinal studies.

[Genetic thrombophilic defects (Factor V Leiden, prothrombin G20210A, MTHFR C677T) in women with recurrent fetal loss].

Maternal thrombophilia (inherited and acquired) has recently been identified as a major cause of thrombembolism, but it may also contribute to adverse pregnancy outcomes and recurrent pregnancy loss. To determine the association of specific inherited thrombophilias and recurrent fetal loss (RFL), three gene mutations (Factor V Leiden, prothrombin G20210A, MTHFR C677T) were investigated. The prevalence of the thrombophilic markers was compared in 156 women with history of fetal loss in different trimester of pregnancy and 80 matched controls. At least one thrombophilic defect was found in 28.2% of total study group women compared with 16.2% in controls (p=0.06; OR-2.02) and in 50% of women with RFL in third trimester (p=0.008; OR-5.15). Factor V Leiden was more common in the group of women with fetal loss in third trimester (37.5%) compared to the controls (6.2%) (p=0.002; OR-9.0). Presence of FVL was associated with a significant increased risk for RFL in second and third trimester (OR-6.25; P<0.001) and significant protection for RFL in first trimester (OR-0.16; P<0.001). Mutation prothrombin G20210A or MTHFR C677T was more common in group of women with fetal loss in first trimester compared to the controls (28.3% vs. 11.2% respectively; p=0.009; OR-3.11). The presence of either of these mutations was associated with no significant increased risk for RFL in first trimester (OR-2.5). Genetic thrombophilic defects are common in women with RFL and are associated with late fetal loss. This association is manifest by FVL rather than total number of defects involved.

[Carriers of thrombophilic factor among women with preeclampsia (preliminary report)].

The aim of this study was to determine whether inherited thrombophilia increases the risk of mild preeclampsia. Twenty five women who developed mild preeclampsia and 49 controls--women with previous uneventful pregnancies, were tested for factor V Leiden, C677T gene variant of methylenetetrahydrofolate reductase (MTHFR), polymorphism 4G/5G in plasminogen activator inhibitor 1 (PAI 1), polymorphism A1/A2 in platelet glycoprotein IIb/Illa (GIPrllb/llla A1/A2). The higher but not significant prevalence of C677T gene variant and polymorphism A1/A2 in women with preeclampsia compared with controls was found: 32% and 12.2%, respectively for cases and controls for both factors, with OR: 3.37 (95% CI 0.883-13.2), p > 0.05. The values of OR and RR for these two thrombophilic factors show that platelet integrin polymorphisms (GIPrIIb/llla A1/A2) and C677T gene variant might be have an important role for development of preeclampsia. The carriage of FVL was with a very small prevalence in women with preeclampsia (8%) as compared to controls (6,1%), with OR: 1.333 (CI 95% 0.143-10.864), p > 0.05. The similar results were found for carriage of polymorphism 4G/5G in PAI-1: gene, respectively 24% u 18.4% in women with preeclampsia as compared to controls, OR: 1.404 (95% CI 0.374-5.14), p > 0.05. The results are not significant, because of the small group of selected patients. Larger case-control study should be executed for the evaluation of impact of inherited thrombophilic factors in the development of mild preeclampsia.

Discrete reduction of type I collagen thermal stability upon oxidation.

The oxidation of acid-soluble calf skin collagen type I caused by metal-dependent free radical generating systems, Fe(II)/H2O2 and Cu(II)/H2O2, was found to bring down in a specific, discrete way the collagen thermal stability, as determined by microcalorimetry and scanning densitometry. Initial oxidation results in splitting of the collagen denaturational transition into two components. Along with the endotherm at 41 degrees C typical for non-oxidized collagen, a second, similarly cooperative endotherm appears at 35 degrees C and increases in enthalpy with the oxidant concentration and exposure time, while the first peak correspondingly decreases. The two transitions at 35 and 41 degrees C were registered by densitometry as stepwise increases of the collagen-specific volume. Further oxidation results in massive collagen destruction manifested as abolishment of both denaturational transitions. The two oxidative systems used produce identical effects on the collagen stability but at higher concentrations of Cu(II) in comparison to Fe(II). The discrete reduction of the protein thermal stability is accompanied by a decrease of the free amino groups, suggestive of an oxidation attack of the side chains of lysine residues. Since the denaturation temperature of collagen shifts from above to below body temperature (41 degrees C-35 degrees C) upon oxidation, it appears important to account for this effect in a context of the possible physiological implications of collagen oxidation.

Advantages of orange-labelled collagen and gelatine as substrates for rapid collagenase activity measurement.

A simple method for the determination of collagenase activity utilising dye-labelled substrates is proposed. It consists in labelling gelatine and bovine Achilles tendon collagen under nondenaturing conditions with the dye active orange GT. As verified with two different enzyme preparations, labelling did not dramatically change the susceptibility to collagenases. The kinetic parameters obtained for dye-labelled collagen and gelatine were compared to those obtained for the hydrolysis of native insoluble collagen (Mandl's method). The method offers the following advantages: it is rapid, reproducible, does not require special equipment and is more specific for collagenases than the widely used azocoll and Mandl methods.