Vitamin K

Vitamin K status


LAS2B869

Contact: Natasja van Schoor

Background
Vitamin K in its reduced form is a cofactor for one enzyme, gamma-glutamylcarboxylase, which post-translationally gamma-carboxylates certain glutamic acid residues in a number of vitamin–dependent (VKD) proteins. Gamma-carboxylation allows VKD proteins to bind calcium, is needed for proper folding of the Gla domain, and facilitates binding of Gla proteins to cell membranes. An example of VKD proteins that are gamma-carboxylated in extrahepatic tissue, i.e. primarily bone, cartilage, and in vascular tissue, is the matrix γ-carboxyglutamate (Gla) protein (MGP). MGP is an important local inhibitor of vascular calcification, which can undergo two post-translational modifications: vitamin K-dependent γ-glutamate carboxylation and serine phosphorylation. While carboxylation is thought to have effects upon binding of calcium-ions, phosphorylation is supposed to affect the cellular release of MGP. Since both modifications can be exerted incompletely, various MGP species can be detected in the circulation.

The dp-ucMGP assay seems to be particularly suited to assess vascular vitamin K status, and may therefore contribute to cardiovascular risk assessment.

Measurements in LASA

Blood collection
Morning blood samples were collected in 2002/03 when subjects were in a non-fasting state and in a sitting position. EDTA plasma samples were stored at −80 °C until determination in 2011. Out of 748 samples, 52 samples did not contain plasma (total n=696). Furthermore, 9 and 37 samples were indicated by VITAK as hemolytic or light hemolytic; 5 and 16 samples were found to be lipaemic or light lipaemic. Statistical analyses should be performed with and without (light) lipaemic or (light) hemolytic samples (n= 67).

Data analyzed: January-March 2011
Sort of samples: EDTA plasma
Amount of samples: 696
Samples reported to be: pM (pico mol/liter) ucMGP and pM cMGP
Samples analyzed in: Duplicate

Measurement procedure & variable information

Methods: dp-ucMGP measurement
The method for dp-ucMGP measurement was a sandwich (dualantibody) ELISA, with the capture antibody directed against the non-phosphorylated MGP sequence 3–15 (mAb-dpMGP; VitaK BV, Maastricht, the Netherlands) and the detecting antibody directed against the uncarboxylated MGP sequence 35–49 (mAbucMGP; VitaK BV). The mAb-ucMGP was biotinylated using the EZ-Link Maleimide PEO2-Biotin kit (ProImmune, Oxford, UK) according to the manufacturer’s instructions. The synthetic peptide Acetyl-SHESMESYELNPF-(AADO)-VQERIRERSKPVHELNREAC-amide, designated as dpMGP3–15-(AADO)-ucMGP35–54 (Pepscan, Lelystad, the Netherlands), was used as standard. The first 13 amino acid residues of this peptide were homologous to the non-phosphorylated MGP sequence 3–15 and the last 20 amino acid residues to the uncarboxylated MGP sequence 35–54. AADO indicates the linker 8-amino-3,6-dioxaoctanoic acid. The mAb-dpMGP was diluted 200-fold in carbonate buffer (33 mM disodiumcarbonate, 66 mM sodiumbicarbonate, pH 9.6) and used for coating of the microtiter plate (100 μl/well). After incubation for 4 hours at room temperature, the remaining binding sites were blocked with 200 μl blocking buffer (2% HNBSA, consisting of HEPES-NaCl buffer [25 mM HEPES, 175 mM NaCl, pH 7.7] and 2% bovine serum albumin [BSA; Sigma, St.Louis, MO, USA]). The plate was incubated overnight at 4ºC and subsequently washed four times with 300 μl washing buffer (0.05% Tween-20 in HEPES-NaCl buffer). Next, 90 μl of citrate plasma sample were diluted in 135 μl 0.2% Protifar (Nutricia, Zoetermeer, the Netherlands) and the dpMGP3–15-(AADO)-ucMGP35–54 peptide was diluted 83,000 fold in 0.2% Protifar; 75 μl of either solution were transferred to the microtiter plate. Subsequently, 25 μl of the biotinylated mAb-ucMGP diluted 750-fold in 0.2% Protifar were added to the plate and the plate was incubated with shaking (150 rpm) overnight at 4ºC. After four washing cycles with washing buffer, the plate was incubated with 100 μl streptavidin-peroxide (Zymed, Breda, the Netherlands) diluted in HEPES-NaCl buffer and 0.5% BSA during 30 minutes (min) at room temperature. The plate was stained with 100 μl of 3,3’,5, 5’tetramethylbenzidine (KLP, Gennep, the Netherlands) during 6–7 min. The staining process was stopped by adding 50 μl of 1.0 M H2SO4 , and the plate was read at 450 nm.

Methods: dp-cMGP measurement
The method for dp-cMGP measurement was also a sandwich ELISA, in which mAb-dpMGP again served as a capture antibody. The detecting antibody was directed against the carboxylated MGP sequence 35–54 (mAb-cMGP; VitaK BV, Maastricht, the Netherlands). The mAb-cMGP was biotinylated using the EZ-Link Maleimide PEO2-Biotin kit (Pierce) according to the manufacturer’s instructions. The standard peptide for the dp-cMGP assay, designated as dpMGP3–15-(AADO)-cMGP35–54 (ProImmune), was a similar peptide as described for the dp-ucMGP assay, in which glutamate was replaced by Gla. In brief, the mAb-dpMGP was diluted 200-fold in carbonate buffer and the mAb-cMGP 1,500 times in 0.2% Protifar. The dpMGP3–15-(AADO)-cMGP35–54 synthetic peptide was diluted 83,000 fold in 0.2% in Protifar. From the standard or citrate plasma dilution (30 μl of citrate plasma in 195 μl of 0.2% Protifar), 25 μl were added to the microtiter plate. The method was otherwise the same as described for the dp-ucMGP assay.


Table 1. Assay characteristics


dp-ucMGP

dp-cMGP

Intra-assay variation (%)

5.6

3.7

Inter-assay variation (%)

9.9

11.5

Lower detection limit (pM)

21

60

Within-day variation (%)

5.5

7.4

Day-to-day variation (%)

8.4

13.1


The range of dp-ucMGP in healthy people is dependent on age and is between 100pM and 600pM, although sometimes higher concentrations are possible. The same holds true for the range of dp-cMGP, which is between 1000pM and 2000pM. Zero values are very rare in a healthy population.

Figures First 2 charts are on dp-ucMGP, last two on dp-cMGP. (pdf)

Availability of data per wave

Numbers per wave

 

B

C

 
2B*

G

 

3B*

All regions

 

 

696

 

 

*  2B=baseline second cohort;
    3B=baseline third cohort

Previous use in LASA
Research of Van den Heuvel et al. (2014) shows that insufficiency of vitamin K, as assessed by high plasma dp-ucMGP concentrations, is associated with an increased risk for cardiovascular disease independent of classical risk factors and vitamin D status.

  • Van den Heuvel, E.G.H.M., Van Schoor , N.M., Lips, P.T.A., Magdeleyns, E.J.P., Deeg, D.J.H., Vermeer, C., Den Heijer, M. (2014). Circulating uncarboxylated matrix Gla protein, a marker of vitamin K status, as a risk factor of cardiovascular disease. Maturitas, 77, 137-141.

Reference

  1. Cranenburg ECM; Koos R; Schurgers LJ; Magdeleyns EJ; Schoonbrood THM ; Landewé RB; Brandenburg VM; Bekers O; Vermeer C. Characterisation and potential diagnostic value of circulating matrix Gla protein (MGP) species. Thromb Haemost 2010; 104: 811–822.