Method For Detection Of Inhibitors Research Articles

Development of an on-line high performance liquid chromatography detection system for human cytochrome P450 1A2 inhibitors in extracts of natural products

An on-line HPLC screening method for detection of inhibitors of human cytochrome P450 1A2 in extracts was developed. HPLC separation of extracts is connected to a continuous methoxyresorufin- O-demethylation (MROD) assay in which recombinant human P450 1A2 converts methoxyresorufin to its fluorescent metabolite resorufin. The system was tested with three P450 1A2 inhibitors, for which minimum detectable amounts (MDA) ranging from 0.7 to 9.5 ng were obtained. Analysis of a kava kava and a basil extract showed that the on-line system is applicable to complex mixtures, since in both extracts, peaks with P450 1A2 inhibiting activity were observed.

Detection of factor VIII inhibitors with the partial thromboplastin time

Variations of the partial thromboplastin time (PTT) were tested to determine the best screening method for detection of inhibitors of factor VIII. Variables tested included the duration of preincubation of a mixture of patient plasma and factor VIII source (normal plasma), the ratio of the patient plasma to the normal plasma, and the duration of incubation of the normal plasma-patient plasma mixture with kaolin- cephalin suspension prior to recalcification. The following conclusions were reached: (1) The PTT performed on a mixture of equal amounts of patient and normal plasma without preincubation of the mixture was inadequate to detect many factor VIII inhibitors. (2) Factor VIII inhibitors of more than 0.5 Bethesda units could be detected if the PTT was performed on a mixture of four parts patient plasma and one part normal plasma, with preincubation of the mixture for 60 min at 37 degrees C. (3) Factor VIII inhibitors as weak as 0.1 Bethesda units could be detected if the PTT was performed on a mixture of four parts patient plasma and one part normal plasma incubated with kaolin- cephalin suspension for 120 min at 37 degrees C before recalcification. The last method may make detection of mild factor VIII inhibitors possible in routine clinical laboratories not equipped to perform the more technically difficult Bethesda inhibitor assays.

Detection of Factor VIII Inhibitors With the Partial Thromboplastin Time

Variations of the partial thromboplastin time (PTT) were tested to determine the best screening method for detection of inhibitors of factor VIII. Variables tested included the duration of preincubation of a mixture of patient plasma and factor VIII source (normal plasma), the ratio of the patient plasma to the normal plasma, and the duration of incubation of the normal plasma-patient plasma mixture with kaolin-cephalin suspension prior to recalcification. The following conclusions were reached: (1) The PTT performed on a mixture of equal amounts of patient and normal plasma without preincubation of the mixture was inadequate to detect many factor VIII inhibitors. (2) Factor VIII inhibitors of more than 0.5 Bethesda units could be detected if the PTT was performed on a mixture of four parts patient plasma and one part normal plasma, with preincubation of the mixture for 60 min at 37 degrees C. (3) Factor VIII inhibitors as weak as 0.1 Bethesda units could be detected if the PTT was performed on a mixture of four parts patient plasma and one part normal plasma incubated with kaolin-cephalin suspension for 120 min at 37 degrees C before recalcification. The last method may make detection of mild factor VIII inhibitors possible in routine clinical laboratories not equipped to perform the more technically difficult Bethesda inhibitor assays.