Nagalase (n-acetylgactosaminidase) is an enzyme that deglycosylates the Gc protein also known as vitamin D binding protein (VDBP). The Gc protein is thereby rendered incapable of conversion into active GcMAF. As a result, the regulation of macrophage activation is prevented.

Activity of α-N-acetylgalactosaminidase (Nagalase) in serum or plasma is determined through a proprietary method consisting of a two step immunocapture assay.

Nagalase is known to inactivate Gc-MAF and his natural precursor Gc-globulin. It is proposed that patients could be treated with substitution therapy with Gc-MAF and Nagalase has been projected as marker (indicator) of effectiveness for the Gc-MAF therapy.

Apparent nagalase activity in serum or plasma is measured kinetically at RED Laboratories through conversion of a fluorogenic substrate in function of time. The test is standardized against a large serum pool of carefully selected healthy persons (normal WBC count, no inflammation, CRP <1mg/L, no clinical history of immune disease of diabetes) the apparent nagalase activity measurement has allowed to establish a normal range of substrate conversion between 0,5 and 0,95 nMol/ml/min for adults.

Our extensive research in this field however revealed that the measurement of the apparent nagalase activity does only reflect the nagalase activity as potential inactivating enzyme activity in serum/plasma for GC-MAF, but it does not reflect the total nagalase activity in serum, because with the measurement through fluorogenic substrate conversion one should take into account the amount of natural substrates present in the serum/plasma which compete for the substrate conversion in our assay. The known natural substrates for Nagalase are (i) Gc-MAF (at very low concentration ng/ml), (ii) Gc-globulin (at high concentration on average 620 mg/L in serum) and (iii) blood group A substance (in extremely high concentrations in total blood due to the amount of RBC, but only in low concentrations in serum or plasma). The amount of natural substrate for nagalase present in tissues is not known for the moment.

This situation leads to the conclusion that the apparent Nagalase activity is not a good biomarker to measure the breakdown of Gc-MAF in the blood of patients and we should taken into account the presence of natural competitors of Nagalase in order to establish the total nagalase content in serum/plasma. The main natural competitor we can measure in blood is the Gc-globulin concentration, which may vary between 150 and 1500 mg/L. The Gc-globulin concentration may affect the Gc-MAF concentration in two ways: first as being the precursor of Gc-MaF affecting the buildup of Gc-MaF through the activating enzyme co-operation on B and T lymphocytes and secondly as a major competitor for Nagalase preventing the breakdown of Gc-MAF.

In line with this, the results for Nagalase testing done at RED Laboratories are reported as a group containing the values for (i) apparent nagalase activity, (ii) Gc-globulin concentration, (iii) corrected Nagalase activity (i.e. effective nagalase activity) in function of the major natural substrate competitor Gc-globulin and (iv) an alternative immunomodulation enzyme for which we have choose Dipeptidyl-peptidase or CD26 present on T and B lymphocytes and macrophages as well as in numerous other cell populations in the body. The normal range values have been established for children, adolescents and adults.

Sample Collection

Testing on plasma: Whole blood must be collected in 1x9ml (or 2×4,5ml) EDTA tubes. Tubes must be centrifuged by nurse/physician within one hour at 3000 rpm for 10 minutes. Plasma has to be aliquoted in new, sterile tubes and frozen. Immediately freeze at least 3 ml of plasma and ship to the lab on dry ice. Sample must be kept frozen at all time.

Testing on Serum: Whole blood must be collected in serum tubes (like BD Vacutainer SST II Advanced tube, yellow cap). Tubes must be centrifuged by nurse/physician at 3000 rpm for 10 minutes after one hour of clotting at room temperature. Serum has to be aliquoted in new, sterile tubes and frozen. Immediately freeze at least 3ml of serum and ship to the lab on dry ice. Sample must be kept frozen at all time. Centrifuged serum tube could be shipped on ambient temperature if its delivery is ensured within 24h hours after the blood sampling.

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