Background: How the vitamin K oxidoreductase (VKORC1) supports vitamin K-dependent protein carboxylation is poorly understood. Results: VKORC1 multimers efficiently perform both reactions that reduce vitamin K, and the inactive monomer in wild type mutant heteromers suppresses reduction. Conclusion: VKORC1 fully reduces vitamin K required for carboxylation. Significance: Multimers are important in VKORC1 mechanism and wild type mutant heteromers impact patients with warfarin resistance.

The anticoagulant warfarin inhibits the vitamin K oxidoreductase (VKORC1), which generates vitamin K hydroquinone (KH) required for the carboxylation and consequent activation of vitamin K-dependent (VKD) proteins. VKORC1 produces KH in 2 reactions: reduction of vitamin K epoxide (KO) to quinone (K), and then KH Our dissection of full reduction vs the individual reactions revealed a surprising mechanism of warfarin inhibition. Warfarin inhibition of KO to K reduction and carboxylation that requires full reduction were compared in wild-type VKORC1 or mutants (Y139H, Y139F) that cause warfarin resistance. Carboxylation was much more strongly inhibited (∼400-fold) than KO reduction (two- to threefold). The K to KH reaction was analyzed using low K concentrations that result from inhibition of KO to K. Carboxylation that required only K to KH reduction was inhibited much less than observed with the KO substrate that requires full VKORC1 reduction (eg, 2.5-fold vs 70-fold, respectively, in cells expressing wild-type VKORC1 and factor IX). The results indicate that warfarin uncouples the 2 reactions that fully reduce KO. Uncoupling was revealed because a second activity, a warfarin-resistant quinone reductase, was not present. In contrast, 293 cells expressing factor IX and this reductase activity showed much less inhibition of carboxylation. This activity therefore appears to cooperate with VKORC1 to accomplish full KO reduction. Cooperation during warfarin therapy would have significant consequences, as VKD proteins function in numerous physiologies in many tissues, but may be poorly carboxylated and dysfunctional if the second activity is not ubiquitously expressed similar to VKORC1.

Retail samples of livers from calf (23), ox (18), lamb (17), pig (15), chicken (16) and turkey (1) were analysed to determine levels of vitamin A (all trans-retinol) and to aid assessment of the effects of using vitamin supplemented compound feedingstuffs for livestock. For comparison, 22 liver samples from lambs reared on diets not containing vitamin-supplemented compound feedingstuffs and four samples of liver from ox which had received supplemented feed but not during the last four months prior to slaughter were also analysed. The chosen method of analysis utilized saponification, solvent extraction and normal-phase high performance liquid chromatography with fluorescence detection. For all species analysed, the levels of vitamin A ranged from 10 to 1100 mg/kg, with all but seven at or below 400 mg/kg. For lamb and ox livers, the mean levels were 310 mg/kg and 200 mg/kg respectively for retail samples. The mean levels were 220 mg/kg (lamb) and 120 mg/kg (ox) in liver samples from animals fed controlled diets. The results are of the same order as those reported over recent years.

γ-Glutamyl carboxylase (GGCX) generates multiple carboxylated Glus (Glas) in vitamin K–dependent (VKD) proteins that are required for their functions. GGCX is processive, remaining bound to VKD proteins throughout multiple Glu carboxylations, and this study reveals the essentiality of processivity to VKD protein function. GGCX mutants (V255M and S300F) whose combined heterozygosity in a patient causes defective clotting and calcification were studied using a novel assay that mimics in vivo carboxylation. Complexes between variant carboxylases and VKD proteins important to hemostasis (factor IX [FIX]) or calcification (matrix Gla protein [MGP]) were reacted in the presence of a challenge VKD protein that could potentially interfere with carboxylation of the VKD protein in the complex. The VKD protein in the complex with wild-type carboxylase was carboxylated before challenge protein carboxylation occurred and became fully carboxylated. In contrast, the V255M mutant carboxylated both forms at the same time and did not completely carboxylate FIX in the complex. S300F carboxylation was poor with both FIX and MGP. Additional studies analyzed FIX- and MGP-derived peptides containing the Gla domain linked to sequences that mediate carboxylase binding. The total amount of carboxylated peptide generated by the V255M mutant was higher than that of wild-type GGCX; however, the individual peptides were partially carboxylated. Analysis of the V255M mutant in FIX HEK293 cells lacking endogenous GGCX revealed poor FIX clotting activity. This study shows that disrupted processivity causes disease and explains the defect in the patient. Kinetic analyses also suggest that disrupted processivity may occur in wild-type carboxylase under some conditions (eg, warfarin therapy or vitamin K deficiency).

We compared a selection of quantitative immunological methods for prostatic acid phosphatase (PAP) with routine colorimetric assays for total and tartrate-labile acid phosphatase and evaluated their relative clinical merits in the differential diagnosis of prostatic carcinoma. We also assessed a wide range of commercial control materials for suitability of use with these methods. Patients studied included 111 cases of prostatic carcinoma, 42 cases of benign prostatic hyperplasia, and 33 controls. The principles of the methods used included determination of enzymatic activity using p-nitrophenyl phosphate, RIA, immunoradiometric, and enzymoimmunometric assays. Performance characteristics for the immunological methods were inferior to manufacturers' precision and specificity claims. We identified control materials that were unsuitable for routine use. Poor discrimination between clinical groups was observed for all methods. Analysis by use of a receiver operator characteristic plot failed to improve this. We conclude that the immunological methods we studied offer no advantages over colorimetric methods in the differential diagnosis of prostatic cancer in symptomatic patients.