Chitotriosidase determination in plasma and in dried blood spots: a comparison using two different substrates in a microplate assay.

BACKGROUND: Chitotriosidase (CT) is a macrophage glycosylhydrolase referred as a biochemical marker on diagnosis and prognosis for Gaucher Disease (GD). The aim of the study was to validate a microplate assay for plasma and dried blood spots on filter paper (DBS) for CT activity determination and to compare CT activity using 4-methylumbelliferyl-b-D-N,N',N''-triacetyl-chitotrioside (4MU-C3) and 4-methylumbelliferyl-deoxychitobiose (4MU-dC2) as substrates. METHODS: Heparinized blood was collected from 12 healthy volunteers (HV) and 14 treated GD patients. Total blood was used to prepare DBS and plasma was obtained by centrifugation. CT activity measurements were done on tube and microplate by a fluorimetric assay using plasma and punches of the DBS samples. RESULTS: The use of 4MU-C3 presented a good correlation among plasma and DBS when analyzing the groups altogether (Pearson=0.76), but the correlation was weaker when we isolated GD patients (Pearson=0.67). The use of 4MU-dC2 increased the correlation among the samples for this group of individuals (Pearson=0.89). CONCLUSION: The possibility of DBS assay facilitates sample transport and storage and CT activity determination on a microplate assay improves laboratory routine. The use of 4MU-dC2 may be more appropriate for GD patients diagnosis on DBS.

Acute stressor-selective effects on homocysteine metabolism and oxidative stress parameters in female rats.

Homocysteine levels are affected by diet factors such as vitamin deficiencies, non-diet factors such as genetic disorders, and stress exposure. Hyperhomocysteinemia has been implicated in several disorders, including cardiovascular disease, depression, schizophrenia, Alzheimer's and Parkinson's disease. Since sex differences play a role both in stress responses and in susceptibility to various diseases, the objective of this study was to evaluate possible alterations in homocysteine metabolism including cysteine, folate, and vitamin B(6), and oxidative stress markers in female rats exposed to different types of acute stress. Female rats were randomly distributed into eight groups according to stress manipulation (restraint, swimming, cold and control) and estrous cycle (diestrus and estrus). In general no significant differences were seen between rats in estrus and diestrus. Restraint stress was the only type of stress that altered homocysteine concentrations (+33% relative to controls). An increase in levels of thiobarbituric acid reactive substances (TBARS) and a decrease in total glutathione (GSHt) concentration were also observed in animals subjected to restraint and swimming stress, suggesting the possibility of oxidative damage. Thus, both the homocysteine results and the oxidative stress data indicated that restraint stress was the most powerful stress manipulation in female rats, as previously observed in male rats. These findings indicate that hormonal and gonadal differences do not interfere with stress responses related to homocysteine metabolism and suggest that putative gender-related differences in homocysteine responses are probably not involved in the differential prevalence of some diseases in human males and females.