Establishment of the Variation of Vitamin K Status According to Vkorc1 Point Mutations Using Rat Models

Vitamin K is crucial for many physiological processes such as coagulation, energy metabolism, and arterial calcification prevention due to its involvement in the activation of several vitamin K-dependent proteins. During this activation, vitamin K is converted into vitamin K epoxide, which must be re-reduced by the VKORC1 enzyme. Various VKORC1 mutations have been described in humans. While these mutations have been widely associated with anticoagulant resistance, their association with a modification of vitamin K status due to a modification of the enzyme efficiency has never been considered. Using animal models with different Vkorc1 mutations receiving a standard diet or a menadione-deficient diet, we investigated this association by measuring different markers of the vitamin K status. Each mutation dramatically affected vitamin K recycling efficiency. This decrease in recycling was associated with a significant alteration of the vitamin K status, even when animals were fed a menadione-enriched diet suggesting a loss of vitamin K from the cycle due to the presence of the Vkorc1 mutation. This change in vitamin K status resulted in clinical modifications in mutated rats only when animals receive a limited vitamin K intake totally consistent with the capacity of each strain to recycle vitamin K.


Introduction
K vitamins are a complex family composed of several similarly fat-soluble molecules having a 2-methyl-1,4-napthoquinone ring in common. The different forms of vitamin K are distinguished according to the length and nature of the carbon chain in position 3 of the naphthoquinone ring [1]. Vitamin K1 or phylloquinone contains a phytyl side chain, which comprises 4 prenyl units. Vitamin K1 is synthesized only by plants, green algae, and some cyanobacteria and is, thus, found mainly in green vegetables. Vitamins K2 or menaquinones (MK) contain an unsaturated aliphatic side chain with a variable number of prenyl units. Depending on the number of prenyl units, vitamins K2 can be divided into different short-chain MK (i.e., mainly menaquinone-4, MK-4) found mainly in meat and fermented foods and long chain MKs mainly of bacterial origin (i.e., MK-7, to . Vitamin K is important for many physiological processes as the cofactor of the gamma-glutamyl carboxylase (GGCX) enzyme [2,3]. This enzyme is involved in the activation of many proteins, called vitamin K-dependent proteins (VKDPs), by catalyzing their γ-glutamyl carboxylation. Vitamin K is, thus, crucial for blood coagulation through the activation of hepatic blood clotting factors II, VII, IX, and X. Vitamin K is also essential for energy metabolism and arterial calcification prevention through the activation of osteocalcin [4] and Matrix Gla Protein [5], respectively, which are both Table 1. Kinetics parameters of VKOR (Vitamin K epoxide reductase) activity and ratio between VKORC1 and VKORC1L1 proteins in different rat tissues according to the Vkorc1 genotype. Results are the mean of four independent determinations with its 95% confidence interval.

Ratio Between VKORC1 and VKORC1L1 Enzymes
Because two different proteins, VKORC1 and VKORC1L1, may be involved in the reduction of vitamin K epoxide in vitamin K quinone, concentrations of both enzymes in liver, testis, kidneys, and lungs were determined by ELISA (Enzyme-linked immunoabsorbent assay) quantitative assays. The results are presented in Table 2. No statistical difference for the ration between VKORC1 and VKORC1L1 was observed between strains. In liver, whatever the strain considered, the concentration of the VKORC1 protein was always much higher than that of the VKORC1L1 protein with no statistical difference between strains except for the Y139F strain for which VKORC1 was 1.5-fold higher than that of the WT strain. In other tissues, concentrations of VKORC1 and VKORC1L1 proteins were comparable with the VKORC1 protein, which is slightly more present than the VKORC1L1 protein Nutrients 2019, 11, 2076 6 of 16 in testis and lung. This is the opposite in the kidney. In the lung, VKORC1L1 expression was 1.5 to 1.9-fold greater in mutant strains when compared to the WT strain.

Vitamin K Status
According to the Vkorc1 Genotype in Rats fed with a Standard Rodent Diet

Vitamin K Concentrations
To evaluate the influence of the Vkorc1 genotype on the vitamin K status, concentrations of vitamin K1, K1OX, MK4, and MK4OX were determined in liver, testis, kidneys, and lungs of rats homozygous for wild type, Y139C, Y139F, or L120Q fed with a standard rodent diet. Vitamin K1 and K1OX were almost undetectable in all the considered tissues, whatever the genotype of rats. Only MK4 and MK4OX were, therefore, quantified. Results are presented in Figure 1. Statistic differences were found between strains only concerning the concentration of MK4.
Whatever the tissues considered, MK4 concentration was systematically higher in rats homozygous for wild type Vkorc1 compared with rats homozygous for Y139F-KORC1 or Y139C-KORC1. In liver, it was 1.5-fold and 2.4-fold higher. In testis, it was 1.7-fold and 2.1-fold higher and, in kidneys, it was 1.9-fold and 2.6-fold higher. In the lung, it was 2.7-fold and 2.8-fold higher when compared with rats homozygous for Y139F-Vkorc1 or Y139C-Vkorc1, respectively.
In liver and testis, MK4 concentration was similar between wild type rats and rats homozygous for L120Q, while it was 1.9-fold higher in lungs and kidneys of wild type rats compared with rats homozygous for L120Q.

Plasma ucOC Concentration
Under-gamma carboxylated osteocalcin (ucOC) and matrix Gla protein (ucMGP) concentration was measured in plasma of rats homozygous for wild type, Y139C, Y139F, or L120Q fed with a standard rodent diet. Results for ucOC are presented in Figure 2. Statistical differences were found only for ucOC concentration between wild type rats and rats homozygous for Y139C and Y139F. UcOC concentration was 6.1-fold and 7.0-fold higher in the plasma of rats homozygous for Y139Fand Y139C-Vkorc1, respectively, compared with wild type rats. A difference between wild type rats and rats homozygous for L120Q was not statistically significant.

Plasma ucOC Concentration
Under-gamma carboxylated osteocalcin (ucOC) and matrix Gla protein (ucMGP) concentration was measured in plasma of rats homozygous for wild type, Y139C, Y139F, or L120Q fed with a standard rodent diet. Results for ucOC are presented in Figure 2. Statistical differences were found only for ucOC concentration between wild type rats and rats homozygous for Y139C and Y139F. UcOC concentration was 6.1-fold and 7.0-fold higher in the plasma of rats homozygous for Y139F-and Y139C-Vkorc1, respectively, compared with wild type rats. A difference between wild type rats and rats homozygous for L120Q was not statistically significant. Figure 2. ucOC concentrations in the plasma of rats homozygous for WT-, L120Q-, Y139C-, or Y139F-Vkorc1 fed with a standard rodent diet. The box extends from the 25th to 75th percentiles and the line corresponds to the mean. Statistical analyses were performed using a Dunn's multiple comparisons test. p < 0.05 was the accepted level of significance. a,b, statistical difference between two groups.

Tissue Calcium Concentration
Calcium concentration was measured in testis, kidneys, lungs, and hearts of rats homozygous for wild type, Y139C, Y139F, or L120Q fed with a standard rodent diet. Results are presented in Figure  3. No difference was observed between strains.

Tissue Calcium Concentration
Calcium concentration was measured in testis, kidneys, lungs, and hearts of rats homozygous for wild type, Y139C, Y139F, or L120Q fed with a standard rodent diet. Results are presented in Figure 3. No difference was observed between strains.

Tissue Calcium Concentration
Calcium concentration was measured in testis, kidneys, lungs, and hearts of rats homozygous for wild type, Y139C, Y139F, or L120Q fed with a standard rodent diet. Results are presented in Figure  3. No difference was observed between strains.   3.3. Evolution of the Vitamin K Status According to the Vkorc1 Genotype in Rats Receiving a Specific Diet-K3 for 12 Days

Vitamin K Concentrations
The evolution of MK4 concentrations in different tissues (i.e., testis, kidneys, and lungs) was determined in rats receiving a vitamin K3 deficient diet for a short period of time from 0 to 12 days. The results are presented in Figure 4. This evolution could not be determined in the liver since this concentration was too low even before the beginning of the deficiency. The evolution of MK4 concentrations in different tissues (i.e., testis, kidneys, and lungs) was determined in rats receiving a vitamin K3 deficient diet for a short period of time from 0 to 12 days. The results are presented in Figure 4. This evolution could not be determined in the liver since this concentration was too low even before the beginning of the deficiency. Results are expressed as the mean of the percentage of four animals compared to wild type rats at day 0 ± standard deviation.
Half-lives of MK4 in the different tissues of the different rat strains were calculated using a nonlinear regression model using equation C = (C0) -kel*t . Results are presented in Table 3. Half-life of MK4 was four-fold longer in testis of the wild type when compared to mutated rats. In kidney, half-lives were similar for all strains. In lung, half-life of MK4 was lower in wild type rats when compared to rats homozygous for L120Q or Y139F.  Half-lives of MK4 in the different tissues of the different rat strains were calculated using a non-linear regression model using equation C = (C0) -kel*t . Results are presented in Table 3. Half-life of MK4 was four-fold longer in testis of the wild type when compared to mutated rats. In kidney, half-lives were similar for all strains. In lung, half-life of MK4 was lower in wild type rats when compared to rats homozygous for L120Q or Y139F.

Prothrombin Time
Consequences of the vitamin K3 deficiency were assessed by the determination of the prothrombin time at day 0, 4, 8, and 12 after the beginning of the vitamin K3 deficiency. Results are presented in Figure 5. Prothrombin time was not modified in rats homozygous for the wild type, Y139C, or Y139F fed with a standard rodent diet. Prothrombin time increased in rats homozygous for L120Q from the eighth day after the beginning of the vitamin K3 deficiency. presented in Figure 5. Prothrombin time was not modified in rats homozygous for the wild type, Y139C, or Y139F fed with a standard rodent diet. Prothrombin time increased in rats homozygous for L120Q from the eighth day after the beginning of the vitamin K3 deficiency.

Plasma ucOC Concentration
Consequences of the vitamin K3 deficiency were also assessed by determining the plasma ucOC concentration at day 0, 4, 8, and 12 after the beginning of the vitamin K3 deficiency. Results are presented in Figure 6. The ucOC concentration remained unchanged during the 12 days of vitamin K3 deficiency in wild type rats, while, in the other strains, ucOC concentration increased rapidly. ucOC concentration reached its maximum 4 days after the beginning of the vitamin K3 deficiency and increased two-fold in rats homozygous for Y139F and Y139C and four-fold in rats homozygous for L120Q.  Figure 6. ucOC concentrations evolution in plasma rats homozygous for WT-, L120Q-, Y139C-, or Y139F-Vkorc1 receiving a specific diet -K3 for 12 days. Results are expressed as the mean of the percentage of 4 animals compared to wild type rats at day 0 ± standard deviation.

Discussion
Anticoagulant-resistant rats due to Vkorc1 gene mutations are good models for mimicking the consequences of human VKORC1 mutations. In humans, mutations of the VKORC1 promoter would decrease VKORC1 expression and some VKORC1 coding mutations would affect its VKOR activity [30]. The consequences of these mutations may, therefore, be different, in terms of activity and/or the consequences on vitamin K status. Using the rat as a model, we have considered these possible differences depending on the nature of the mutation. For this, we used four different rat strains, the OFA-Sprague Dawley rats, and three strains of introgressed rats homozygous for mutations L120Q, Y139F, or Y139C, with the amino acid 139 being located near the catalytic site, and the amino acid 120

Plasma ucOC Concentration
Consequences of the vitamin K3 deficiency were also assessed by determining the plasma ucOC concentration at day 0, 4, 8, and 12 after the beginning of the vitamin K3 deficiency. Results are presented in Figure 6. The ucOC concentration remained unchanged during the 12 days of vitamin K3 deficiency in wild type rats, while, in the other strains, ucOC concentration increased rapidly. ucOC concentration reached its maximum 4 days after the beginning of the vitamin K3 deficiency and increased two-fold in rats homozygous for Y139F and Y139C and four-fold in rats homozygous for L120Q.
Nutrients 2019, 11, x FOR PEER REVIEW 10 of 16 presented in Figure 5. Prothrombin time was not modified in rats homozygous for the wild type, Y139C, or Y139F fed with a standard rodent diet. Prothrombin time increased in rats homozygous for L120Q from the eighth day after the beginning of the vitamin K3 deficiency.

Plasma ucOC Concentration
Consequences of the vitamin K3 deficiency were also assessed by determining the plasma ucOC concentration at day 0, 4, 8, and 12 after the beginning of the vitamin K3 deficiency. Results are presented in Figure 6. The ucOC concentration remained unchanged during the 12 days of vitamin K3 deficiency in wild type rats, while, in the other strains, ucOC concentration increased rapidly. ucOC concentration reached its maximum 4 days after the beginning of the vitamin K3 deficiency and increased two-fold in rats homozygous for Y139F and Y139C and four-fold in rats homozygous for L120Q.  Figure 6. ucOC concentrations evolution in plasma rats homozygous for WT-, L120Q-, Y139C-, or Y139F-Vkorc1 receiving a specific diet -K3 for 12 days. Results are expressed as the mean of the percentage of 4 animals compared to wild type rats at day 0 ± standard deviation.

Discussion
Anticoagulant-resistant rats due to Vkorc1 gene mutations are good models for mimicking the consequences of human VKORC1 mutations. In humans, mutations of the VKORC1 promoter would decrease VKORC1 expression and some VKORC1 coding mutations would affect its VKOR activity [30]. The consequences of these mutations may, therefore, be different, in terms of activity and/or the consequences on vitamin K status. Using the rat as a model, we have considered these possible differences depending on the nature of the mutation. For this, we used four different rat strains, the OFA-Sprague Dawley rats, and three strains of introgressed rats homozygous for mutations L120Q, Y139F, or Y139C, with the amino acid 139 being located near the catalytic site, and the amino acid 120 Figure 6. ucOC concentrations evolution in plasma rats homozygous for WT-, L120Q-, Y139C-, or Y139F-Vkorc1 receiving a specific diet -K3 for 12 days. Results are expressed as the mean of the percentage of 4 animals compared to wild type rats at day 0 ± standard deviation.

Discussion
Anticoagulant-resistant rats due to Vkorc1 gene mutations are good models for mimicking the consequences of human VKORC1 mutations. In humans, mutations of the VKORC1 promoter would decrease VKORC1 expression and some VKORC1 coding mutations would affect its VKOR activity [30]. The consequences of these mutations may, therefore, be different, in terms of activity and/or the consequences on vitamin K status. Using the rat as a model, we have considered these possible differences depending on the nature of the mutation. For this, we used four different rat strains, the OFA-Sprague Dawley rats, and three strains of introgressed rats homozygous for mutations L120Q, Y139F, or Y139C, with the amino acid 139 being located near the catalytic site, and the amino acid 120 being further away from the latter [31]. These strains were derived from the OFA-Sprague Dawley rat in which the mutated Vkorc1 gene was introduced with less than 0.0001% of the initial genome of the wild brown rat. We can, thus, legitimately affect the phenotypic modifications to the Vkorc1 mutation.
We first assessed the impact of the mutation on the ability of each tissue to recycle vitamin K. For this, we measured VKOR activity from liver, kidney, lung, and testicular microsomes. VKORC1 is localized in the membrane of the endoplasmic reticulum [2]. The liver is responsible for the activation of factors II, VII, IX, and X {2]. The kidney, lung, and testis contain high amounts of vitamin K [32] and calcify strongly in the absence of vitamin K [23]. Regardless of the tissue, microsomal VKOR activity is always greatly decreased when VKORC1 is mutated, regardless of the position (i.e., amino acid 120 or amino acid 139) and nature (whether amino acid 139 is replaced by phenylalalanine or cysteine) of the mutation. Affinity for vitamin K does not seem to be modified (Km unchanged), but velocity of VKOR activity is systematically greatly reduced (by a factor of 3 to 6 depending on the tissue and the mutation). This loss of velocity may be due to a malfunction of VKORC1 due to mutations and/or a decrease in the VKORC1 expression level because of instability of the mutated proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung.
The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38].
Surprisingly, while all rats have always received the same standard diet supplemented with menadione, the vitamin K status of rats homozygous for Y139C and Y139F mutations is strongly altered with a sharp decrease (by a factor of two to four) in MK4 concentrations in all tissues considered in this study and a significant increase in plasma ucOC concentration (by a factor of almost 7). The "vitamin K" status of rats homozygous for the L120Q mutation can be considered an intermediate between the wild type rat and the rats homozygous for the Y139C or Y139F mutations (Table 4). It is surprising to observe such a difference in vitamin K status in rats receiving the same diet supplemented with menadione, even if they have Vkorc1 mutations. This decrease is even more surprising in the liver, which is the first organ to capture vitamin K from food, where the intake is the same for all strains of rats. Decreasing the recycling efficiency of vitamin K should eventually lead to a decrease in MK4 concentration, but an increase in MK4OX concentration. This is not observed. These results suggest a loss of vitamin K induced by the mutation. The metabolism of vitamin K is still largely unknown. Metabolism of vitamin K by ω-hydroxylation [39,40] with involvement of cytochromes 4F2 and 4F11 in humans has been described. The substrates of cytochromes 4F2 and 4F11 have not been characterized. In rats, cytochromes metabolizing vitamin K have not been identified. Metabolism of vitamin K in its epoxide form by cytochromes might be more efficient than in its quinone form. Since recycling of vitamin K epoxide is less efficient in mutated rats, the elimination of vitamin K could be, thus, increased via greater cytochrome P450 dependent metabolism of vitamin K epoxide. Nevertheless, the difference in status observed between rats homozygous for the L120Q mutation and rats homozygous for the Y139F and Y139C mutations is surprising, whereas the recycling efficiency of the different tissues is similar, or even lower, in rats homozygous for the L120Q mutation (the efficiency that corresponds to Vmax/Km is systematically the lowest). The "vitamin K" status of rats homozygous for the Y139F or Y139C mutations could be aggravated by a modification of the catalysis mediated by VKORC1 due to the mutation. It has been shown that the replacement of tyrosine 139 by cysteine, and, to a lesser extent by phenylalanine, allowed VKORC1 to catalyze a 3-hydroxylation activity, in addition to its activity of reduction [33,41]. The production of 3-hydroxyvitamin K constitutes an additional pathway of elimination of vitamin K and, thus, precludes the recycling of the vitamin by increasing the vitamin K requirements in these rats. Wild type VKORC1 and VKORC1 mutated at position 120 do not produce this 3-hydroxyvitamin K [33]. appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4 status was evaluated by determining tissue vitamin K concentrations, measurement of plasma concentration, and determination of tissue calcium levels. Among the K vitamins, only MK measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplem with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standar being limited and provided by the cereals incorporated). The latter received virtually no vitam and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectabl tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, the considered a good marker of overall vitamin K status. Tissue calcium concentrations have als shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit calcification [23,37,38]. s their sole source of vitamin K (the amounts of vitamin K1 in the standard feed provided by the cereals incorporated). The latter received virtually no vitamin K1 no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat in is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the n K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, marker of overall vitamin K status. Tissue calcium concentrations have also been ively correlated with the vitamin K status. In the absence of vitamin K, MGP, which K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue ,38]. mary of the results obtained for each strain compared to the WT strain; , decrease mpared to WT strain; , increase for WT strain; =, similar with WT strain.

VKOR ACTIVITY
In liver expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung. The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung. The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung. The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung. The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung. The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung. The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung. The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung. The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung. The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung. The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung. The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung. The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung. The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung. The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. mutations and/or a decrease in the VKORC1 expression level because of instability of the mutated proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung. The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. mutations and/or a decrease in the VKORC1 expression level because of instability of the mutated proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung. The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38].

VKORC1 CONCENTRATION
In liver = expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung. The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38].  VKOR activity is always greatly decreased when VKORC1 is mutated, regardless of the position (i.e., amino acid 120 or amino acid 139) and nature (whether amino acid 139 is replaced by phenylalalanine or cysteine) of the mutation. Affinity for vitamin K does not seem to be modified (Km unchanged), but velocity of VKOR activity is systematically greatly reduced (by a factor of 3 to 6 depending on the tissue and the mutation). This loss of velocity may be due to a malfunction of VKORC1 due to mutations and/or a decrease in the VKORC1 expression level because of instability of the mutated proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung.
The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. VKOR activity is always greatly decreased when VKORC1 is mutated, regardless of the position (i.e., amino acid 120 or amino acid 139) and nature (whether amino acid 139 is replaced by phenylalalanine or cysteine) of the mutation. Affinity for vitamin K does not seem to be modified (Km unchanged), but velocity of VKOR activity is systematically greatly reduced (by a factor of 3 to 6 depending on the tissue and the mutation). This loss of velocity may be due to a malfunction of VKORC1 due to mutations and/or a decrease in the VKORC1 expression level because of instability of the mutated proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung. The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. VKOR activity is always greatly decreased when VKORC1 is mutated, regardless of the position (i.e., amino acid 120 or amino acid 139) and nature (whether amino acid 139 is replaced by phenylalalanine or cysteine) of the mutation. Affinity for vitamin K does not seem to be modified (Km unchanged), but velocity of VKOR activity is systematically greatly reduced (by a factor of 3 to 6 depending on the tissue and the mutation). This loss of velocity may be due to a malfunction of VKORC1 due to mutations and/or a decrease in the VKORC1 expression level because of instability of the mutated proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung. The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38].

MK4 CONCENTRATION
In liver = the wild brown rat. We can, thus, legitimately affect the phenotypic modifications to the Vkorc1 mutation. We first assessed the impact of the mutation on the ability of each tissue to recycle vitamin K. For this, we measured VKOR activity from liver, kidney, lung, and testicular microsomes. VKORC1 is localized in the membrane of the endoplasmic reticulum [2]. The liver is responsible for the activation of factors II, VII, IX, and X {2]. The kidney, lung, and testis contain high amounts of vitamin K [32] and calcify strongly in the absence of vitamin K [23]. Regardless of the tissue, microsomal VKOR activity is always greatly decreased when VKORC1 is mutated, regardless of the position (i.e., amino acid 120 or amino acid 139) and nature (whether amino acid 139 is replaced by phenylalalanine or cysteine) of the mutation. Affinity for vitamin K does not seem to be modified (Km unchanged), but velocity of VKOR activity is systematically greatly reduced (by a factor of 3 to 6 depending on the tissue and the mutation). This loss of velocity may be due to a malfunction of VKORC1 due to mutations and/or a decrease in the VKORC1 expression level because of instability of the mutated proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung.
The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. the wild brown rat. We can, thus, legitimately affect the phenotypic modifications to the Vkorc1 mutation. We first assessed the impact of the mutation on the ability of each tissue to recycle vitamin K. For this, we measured VKOR activity from liver, kidney, lung, and testicular microsomes. VKORC1 is localized in the membrane of the endoplasmic reticulum [2]. The liver is responsible for the activation of factors II, VII, IX, and X {2]. The kidney, lung, and testis contain high amounts of vitamin K [32] and calcify strongly in the absence of vitamin K [23]. Regardless of the tissue, microsomal VKOR activity is always greatly decreased when VKORC1 is mutated, regardless of the position (i.e., amino acid 120 or amino acid 139) and nature (whether amino acid 139 is replaced by phenylalalanine or cysteine) of the mutation. Affinity for vitamin K does not seem to be modified (Km unchanged), but velocity of VKOR activity is systematically greatly reduced (by a factor of 3 to 6 depending on the tissue and the mutation). This loss of velocity may be due to a malfunction of VKORC1 due to mutations and/or a decrease in the VKORC1 expression level because of instability of the mutated proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung.
The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. the wild brown rat. We can, thus, legitimately affect the phenotypic modifications to the Vkorc1 mutation. We first assessed the impact of the mutation on the ability of each tissue to recycle vitamin K. For this, we measured VKOR activity from liver, kidney, lung, and testicular microsomes. VKORC1 is localized in the membrane of the endoplasmic reticulum [2]. The liver is responsible for the activation of factors II, VII, IX, and X {2]. The kidney, lung, and testis contain high amounts of vitamin K [32] and calcify strongly in the absence of vitamin K [23]. Regardless of the tissue, microsomal VKOR activity is always greatly decreased when VKORC1 is mutated, regardless of the position (i.e., amino acid 120 or amino acid 139) and nature (whether amino acid 139 is replaced by phenylalalanine or cysteine) of the mutation. Affinity for vitamin K does not seem to be modified (Km unchanged), but velocity of VKOR activity is systematically greatly reduced (by a factor of 3 to 6 depending on the tissue and the mutation). This loss of velocity may be due to a malfunction of VKORC1 due to mutations and/or a decrease in the VKORC1 expression level because of instability of the mutated proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung.
The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. In testis = rat in which the mutated Vkorc1 gene was introduced with less than 0.0001% of the initial genome of the wild brown rat. We can, thus, legitimately affect the phenotypic modifications to the Vkorc1 mutation. We first assessed the impact of the mutation on the ability of each tissue to recycle vitamin K. For this, we measured VKOR activity from liver, kidney, lung, and testicular microsomes. VKORC1 is localized in the membrane of the endoplasmic reticulum [2]. The liver is responsible for the activation of factors II, VII, IX, and X {2]. The kidney, lung, and testis contain high amounts of vitamin K [32] and calcify strongly in the absence of vitamin K [23]. Regardless of the tissue, microsomal VKOR activity is always greatly decreased when VKORC1 is mutated, regardless of the position (i.e., amino acid 120 or amino acid 139) and nature (whether amino acid 139 is replaced by phenylalalanine or cysteine) of the mutation. Affinity for vitamin K does not seem to be modified (Km unchanged), but velocity of VKOR activity is systematically greatly reduced (by a factor of 3 to 6 depending on the tissue and the mutation). This loss of velocity may be due to a malfunction of VKORC1 due to mutations and/or a decrease in the VKORC1 expression level because of instability of the mutated proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung.
The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. rat in which the mutated Vkorc1 gene was introduced with less than 0.0001% of the initial genome of the wild brown rat. We can, thus, legitimately affect the phenotypic modifications to the Vkorc1 mutation. We first assessed the impact of the mutation on the ability of each tissue to recycle vitamin K. For this, we measured VKOR activity from liver, kidney, lung, and testicular microsomes. VKORC1 is localized in the membrane of the endoplasmic reticulum [2]. The liver is responsible for the activation of factors II, VII, IX, and X {2]. The kidney, lung, and testis contain high amounts of vitamin K [32] and calcify strongly in the absence of vitamin K [23]. Regardless of the tissue, microsomal VKOR activity is always greatly decreased when VKORC1 is mutated, regardless of the position (i.e., amino acid 120 or amino acid 139) and nature (whether amino acid 139 is replaced by phenylalalanine or cysteine) of the mutation. Affinity for vitamin K does not seem to be modified (Km unchanged), but velocity of VKOR activity is systematically greatly reduced (by a factor of 3 to 6 depending on the tissue and the mutation). This loss of velocity may be due to a malfunction of VKORC1 due to mutations and/or a decrease in the VKORC1 expression level because of instability of the mutated proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung.
The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. rat in which the mutated Vkorc1 gene was introduced with less than 0.0001% of the initial genome of the wild brown rat. We can, thus, legitimately affect the phenotypic modifications to the Vkorc1 mutation. We first assessed the impact of the mutation on the ability of each tissue to recycle vitamin K. For this, we measured VKOR activity from liver, kidney, lung, and testicular microsomes. VKORC1 is localized in the membrane of the endoplasmic reticulum [2]. The liver is responsible for the activation of factors II, VII, IX, and X {2]. The kidney, lung, and testis contain high amounts of vitamin K [32] and calcify strongly in the absence of vitamin K [23]. Regardless of the tissue, microsomal VKOR activity is always greatly decreased when VKORC1 is mutated, regardless of the position (i.e., amino acid 120 or amino acid 139) and nature (whether amino acid 139 is replaced by phenylalalanine or cysteine) of the mutation. Affinity for vitamin K does not seem to be modified (Km unchanged), but velocity of VKOR activity is systematically greatly reduced (by a factor of 3 to 6 depending on the tissue and the mutation). This loss of velocity may be due to a malfunction of VKORC1 due to mutations and/or a decrease in the VKORC1 expression level because of instability of the mutated proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung.
The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. being further away from the latter [31]. These strains were derived from the OFA-Sprague Dawley rat in which the mutated Vkorc1 gene was introduced with less than 0.0001% of the initial genome of the wild brown rat. We can, thus, legitimately affect the phenotypic modifications to the Vkorc1 mutation. We first assessed the impact of the mutation on the ability of each tissue to recycle vitamin K. For this, we measured VKOR activity from liver, kidney, lung, and testicular microsomes. VKORC1 is localized in the membrane of the endoplasmic reticulum [2]. The liver is responsible for the activation of factors II, VII, IX, and X {2]. The kidney, lung, and testis contain high amounts of vitamin K [32] and calcify strongly in the absence of vitamin K [23]. Regardless of the tissue, microsomal VKOR activity is always greatly decreased when VKORC1 is mutated, regardless of the position (i.e., amino acid 120 or amino acid 139) and nature (whether amino acid 139 is replaced by phenylalalanine or cysteine) of the mutation. Affinity for vitamin K does not seem to be modified (Km unchanged), but velocity of VKOR activity is systematically greatly reduced (by a factor of 3 to 6 depending on the tissue and the mutation). This loss of velocity may be due to a malfunction of VKORC1 due to mutations and/or a decrease in the VKORC1 expression level because of instability of the mutated proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung.
The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. being further away from the latter [31]. These strains were derived from the OFA-Sprague Dawley rat in which the mutated Vkorc1 gene was introduced with less than 0.0001% of the initial genome of the wild brown rat. We can, thus, legitimately affect the phenotypic modifications to the Vkorc1 mutation. We first assessed the impact of the mutation on the ability of each tissue to recycle vitamin K. For this, we measured VKOR activity from liver, kidney, lung, and testicular microsomes. VKORC1 is localized in the membrane of the endoplasmic reticulum [2]. The liver is responsible for the activation of factors II, VII, IX, and X {2]. The kidney, lung, and testis contain high amounts of vitamin K [32] and calcify strongly in the absence of vitamin K [23]. Regardless of the tissue, microsomal VKOR activity is always greatly decreased when VKORC1 is mutated, regardless of the position (i.e., amino acid 120 or amino acid 139) and nature (whether amino acid 139 is replaced by phenylalalanine or cysteine) of the mutation. Affinity for vitamin K does not seem to be modified (Km unchanged), but velocity of VKOR activity is systematically greatly reduced (by a factor of 3 to 6 depending on the tissue and the mutation). This loss of velocity may be due to a malfunction of VKORC1 due to mutations and/or a decrease in the VKORC1 expression level because of instability of the mutated proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung.
The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. being further away from the latter [31]. These strains were derived from the OFA-Sprague Dawley rat in which the mutated Vkorc1 gene was introduced with less than 0.0001% of the initial genome of the wild brown rat. We can, thus, legitimately affect the phenotypic modifications to the Vkorc1 mutation. We first assessed the impact of the mutation on the ability of each tissue to recycle vitamin K. For this, we measured VKOR activity from liver, kidney, lung, and testicular microsomes. VKORC1 is localized in the membrane of the endoplasmic reticulum [2]. The liver is responsible for the activation of factors II, VII, IX, and X {2]. The kidney, lung, and testis contain high amounts of vitamin K [32] and calcify strongly in the absence of vitamin K [23]. Regardless of the tissue, microsomal VKOR activity is always greatly decreased when VKORC1 is mutated, regardless of the position (i.e., amino acid 120 or amino acid 139) and nature (whether amino acid 139 is replaced by phenylalalanine or cysteine) of the mutation. Affinity for vitamin K does not seem to be modified (Km unchanged), but velocity of VKOR activity is systematically greatly reduced (by a factor of 3 to 6 depending on the tissue and the mutation). This loss of velocity may be due to a malfunction of VKORC1 due to mutations and/or a decrease in the VKORC1 expression level because of instability of the mutated proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung.
The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. being further away from the latter [31]. These strains were derived from the OFA-Sprague Dawley rat in which the mutated Vkorc1 gene was introduced with less than 0.0001% of the initial genome of the wild brown rat. We can, thus, legitimately affect the phenotypic modifications to the Vkorc1 mutation. We first assessed the impact of the mutation on the ability of each tissue to recycle vitamin K. For this, we measured VKOR activity from liver, kidney, lung, and testicular microsomes. VKORC1 is localized in the membrane of the endoplasmic reticulum [2]. The liver is responsible for the activation of factors II, VII, IX, and X {2]. The kidney, lung, and testis contain high amounts of vitamin K [32] and calcify strongly in the absence of vitamin K [23]. Regardless of the tissue, microsomal VKOR activity is always greatly decreased when VKORC1 is mutated, regardless of the position (i.e., amino acid 120 or amino acid 139) and nature (whether amino acid 139 is replaced by phenylalalanine or cysteine) of the mutation. Affinity for vitamin K does not seem to be modified (Km unchanged), but velocity of VKOR activity is systematically greatly reduced (by a factor of 3 to 6 depending on the tissue and the mutation). This loss of velocity may be due to a malfunction of VKORC1 due to mutations and/or a decrease in the VKORC1 expression level because of instability of the mutated proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung.
The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. being further away from the latter [31]. These strains were derived from the OFA-Sprague Dawley rat in which the mutated Vkorc1 gene was introduced with less than 0.0001% of the initial genome of the wild brown rat. We can, thus, legitimately affect the phenotypic modifications to the Vkorc1 mutation. We first assessed the impact of the mutation on the ability of each tissue to recycle vitamin K. For this, we measured VKOR activity from liver, kidney, lung, and testicular microsomes. VKORC1 is localized in the membrane of the endoplasmic reticulum [2]. The liver is responsible for the activation of factors II, VII, IX, and X {2]. The kidney, lung, and testis contain high amounts of vitamin K [32] and calcify strongly in the absence of vitamin K [23]. Regardless of the tissue, microsomal VKOR activity is always greatly decreased when VKORC1 is mutated, regardless of the position (i.e., amino acid 120 or amino acid 139) and nature (whether amino acid 139 is replaced by phenylalalanine or cysteine) of the mutation. Affinity for vitamin K does not seem to be modified (Km unchanged), but velocity of VKOR activity is systematically greatly reduced (by a factor of 3 to 6 depending on the tissue and the mutation). This loss of velocity may be due to a malfunction of VKORC1 due to mutations and/or a decrease in the VKORC1 expression level because of instability of the mutated proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung.
The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. being further away from the latter [31]. These strains were derived from the OFA-Sprague Dawley rat in which the mutated Vkorc1 gene was introduced with less than 0.0001% of the initial genome of the wild brown rat. We can, thus, legitimately affect the phenotypic modifications to the Vkorc1 mutation. We first assessed the impact of the mutation on the ability of each tissue to recycle vitamin K. For this, we measured VKOR activity from liver, kidney, lung, and testicular microsomes. VKORC1 is localized in the membrane of the endoplasmic reticulum [2]. The liver is responsible for the activation of factors II, VII, IX, and X {2]. The kidney, lung, and testis contain high amounts of vitamin K [32] and calcify strongly in the absence of vitamin K [23]. Regardless of the tissue, microsomal VKOR activity is always greatly decreased when VKORC1 is mutated, regardless of the position (i.e., amino acid 120 or amino acid 139) and nature (whether amino acid 139 is replaced by phenylalalanine or cysteine) of the mutation. Affinity for vitamin K does not seem to be modified (Km unchanged), but velocity of VKOR activity is systematically greatly reduced (by a factor of 3 to 6 depending on the tissue and the mutation). This loss of velocity may be due to a malfunction of VKORC1 due to mutations and/or a decrease in the VKORC1 expression level because of instability of the mutated proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung.
The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. being further away from the latter [31]. These strains were derived from the OFA-Sprague Dawley rat in which the mutated Vkorc1 gene was introduced with less than 0.0001% of the initial genome of the wild brown rat. We can, thus, legitimately affect the phenotypic modifications to the Vkorc1 mutation.
We first assessed the impact of the mutation on the ability of each tissue to recycle vitamin K. For this, we measured VKOR activity from liver, kidney, lung, and testicular microsomes. VKORC1 is localized in the membrane of the endoplasmic reticulum [2]. The liver is responsible for the activation of factors II, VII, IX, and X {2]. The kidney, lung, and testis contain high amounts of vitamin K [32] and calcify strongly in the absence of vitamin K [23]. Regardless of the tissue, microsomal VKOR activity is always greatly decreased when VKORC1 is mutated, regardless of the position (i.e., amino acid 120 or amino acid 139) and nature (whether amino acid 139 is replaced by phenylalalanine or cysteine) of the mutation. Affinity for vitamin K does not seem to be modified (Km unchanged), but velocity of VKOR activity is systematically greatly reduced (by a factor of 3 to 6 depending on the tissue and the mutation). This loss of velocity may be due to a malfunction of VKORC1 due to mutations and/or a decrease in the VKORC1 expression level because of instability of the mutated proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung.
The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. being further away from the latter [31]. These strains were derived from the OFA-Sprague Dawley rat in which the mutated Vkorc1 gene was introduced with less than 0.0001% of the initial genome of the wild brown rat. We can, thus, legitimately affect the phenotypic modifications to the Vkorc1 mutation.
We first assessed the impact of the mutation on the ability of each tissue to recycle vitamin K. For this, we measured VKOR activity from liver, kidney, lung, and testicular microsomes. VKORC1 is localized in the membrane of the endoplasmic reticulum [2]. The liver is responsible for the activation of factors II, VII, IX, and X {2]. The kidney, lung, and testis contain high amounts of vitamin K [32] and calcify strongly in the absence of vitamin K [23]. Regardless of the tissue, microsomal VKOR activity is always greatly decreased when VKORC1 is mutated, regardless of the position (i.e., amino acid 120 or amino acid 139) and nature (whether amino acid 139 is replaced by phenylalalanine or cysteine) of the mutation. Affinity for vitamin K does not seem to be modified (Km unchanged), but velocity of VKOR activity is systematically greatly reduced (by a factor of 3 to 6 depending on the tissue and the mutation). This loss of velocity may be due to a malfunction of VKORC1 due to mutations and/or a decrease in the VKORC1 expression level because of instability of the mutated proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung.
The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. being further away from the latter [31]. These strains were derived from the OFA-Sprague Dawley rat in which the mutated Vkorc1 gene was introduced with less than 0.0001% of the initial genome of the wild brown rat. We can, thus, legitimately affect the phenotypic modifications to the Vkorc1 mutation. We first assessed the impact of the mutation on the ability of each tissue to recycle vitamin K. For this, we measured VKOR activity from liver, kidney, lung, and testicular microsomes. VKORC1 is localized in the membrane of the endoplasmic reticulum [2]. The liver is responsible for the activation of factors II, VII, IX, and X {2]. The kidney, lung, and testis contain high amounts of vitamin K [32] and calcify strongly in the absence of vitamin K [23]. Regardless of the tissue, microsomal VKOR activity is always greatly decreased when VKORC1 is mutated, regardless of the position (i.e., amino acid 120 or amino acid 139) and nature (whether amino acid 139 is replaced by phenylalalanine or cysteine) of the mutation. Affinity for vitamin K does not seem to be modified (Km unchanged), but velocity of VKOR activity is systematically greatly reduced (by a factor of 3 to 6 depending on the tissue and the mutation). This loss of velocity may be due to a malfunction of VKORC1 due to mutations and/or a decrease in the VKORC1 expression level because of instability of the mutated proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung.
The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. being further away from the latter [31]. These strains were derived from the OFA-Sprague Dawley rat in which the mutated Vkorc1 gene was introduced with less than 0.0001% of the initial genome of the wild brown rat. We can, thus, legitimately affect the phenotypic modifications to the Vkorc1 mutation. We first assessed the impact of the mutation on the ability of each tissue to recycle vitamin K. For this, we measured VKOR activity from liver, kidney, lung, and testicular microsomes. VKORC1 is localized in the membrane of the endoplasmic reticulum [2]. The liver is responsible for the activation of factors II, VII, IX, and X {2]. The kidney, lung, and testis contain high amounts of vitamin K [32] and calcify strongly in the absence of vitamin K [23]. Regardless of the tissue, microsomal VKOR activity is always greatly decreased when VKORC1 is mutated, regardless of the position (i.e., amino acid 120 or amino acid 139) and nature (whether amino acid 139 is replaced by phenylalalanine or cysteine) of the mutation. Affinity for vitamin K does not seem to be modified (Km unchanged), but velocity of VKOR activity is systematically greatly reduced (by a factor of 3 to 6 depending on the tissue and the mutation). This loss of velocity may be due to a malfunction of VKORC1 due to mutations and/or a decrease in the VKORC1 expression level because of instability of the mutated proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung.
The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. the wild brown rat. We can, thus, legitimately affect the phenotypic modifications to the Vkorc1 mutation. We first assessed the impact of the mutation on the ability of each tissue to recycle vitamin K. For this, we measured VKOR activity from liver, kidney, lung, and testicular microsomes. VKORC1 is localized in the membrane of the endoplasmic reticulum [2]. The liver is responsible for the activation of factors II, VII, IX, and X {2]. The kidney, lung, and testis contain high amounts of vitamin K [32] and calcify strongly in the absence of vitamin K [23]. Regardless of the tissue, microsomal VKOR activity is always greatly decreased when VKORC1 is mutated, regardless of the position (i.e., amino acid 120 or amino acid 139) and nature (whether amino acid 139 is replaced by phenylalalanine or cysteine) of the mutation. Affinity for vitamin K does not seem to be modified (Km unchanged), but velocity of VKOR activity is systematically greatly reduced (by a factor of 3 to 6 depending on the tissue and the mutation). This loss of velocity may be due to a malfunction of VKORC1 due to mutations and/or a decrease in the VKORC1 expression level because of instability of the mutated proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung.
The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. the wild brown rat. We can, thus, legitimately affect the phenotypic modifications to the Vkorc1 mutation. We first assessed the impact of the mutation on the ability of each tissue to recycle vitamin K. For this, we measured VKOR activity from liver, kidney, lung, and testicular microsomes. VKORC1 is localized in the membrane of the endoplasmic reticulum [2]. The liver is responsible for the activation of factors II, VII, IX, and X {2]. The kidney, lung, and testis contain high amounts of vitamin K [32] and calcify strongly in the absence of vitamin K [23]. Regardless of the tissue, microsomal VKOR activity is always greatly decreased when VKORC1 is mutated, regardless of the position (i.e., amino acid 120 or amino acid 139) and nature (whether amino acid 139 is replaced by phenylalalanine or cysteine) of the mutation. Affinity for vitamin K does not seem to be modified (Km unchanged), but velocity of VKOR activity is systematically greatly reduced (by a factor of 3 to 6 depending on the tissue and the mutation). This loss of velocity may be due to a malfunction of VKORC1 due to mutations and/or a decrease in the VKORC1 expression level because of instability of the mutated proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung.
The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38]. the wild brown rat. We can, thus, legitimately affect the phenotypic modifications to the Vkorc1 mutation. We first assessed the impact of the mutation on the ability of each tissue to recycle vitamin K. For this, we measured VKOR activity from liver, kidney, lung, and testicular microsomes. VKORC1 is localized in the membrane of the endoplasmic reticulum [2]. The liver is responsible for the activation of factors II, VII, IX, and X {2]. The kidney, lung, and testis contain high amounts of vitamin K [32] and calcify strongly in the absence of vitamin K [23]. Regardless of the tissue, microsomal VKOR activity is always greatly decreased when VKORC1 is mutated, regardless of the position (i.e., amino acid 120 or amino acid 139) and nature (whether amino acid 139 is replaced by phenylalalanine or cysteine) of the mutation. Affinity for vitamin K does not seem to be modified (Km unchanged), but velocity of VKOR activity is systematically greatly reduced (by a factor of 3 to 6 depending on the tissue and the mutation). This loss of velocity may be due to a malfunction of VKORC1 due to mutations and/or a decrease in the VKORC1 expression level because of instability of the mutated proteins. The malfunction is certain. The characterization of the corresponding recombinant proteins has demonstrated this loss of velocity [33]. On the other hand, this study shows that the VKORC1 expression level remains unchanged despite the presence of mutations L120Q, Y139F, or Y139C regardless of the tissue, except in the liver of the Y139F strain where VKORC1 is slightly expressed. Expression of VKORC1L1, which is another enzyme that is able to catalyze VKOR activity [34] and, thus, compensate for loss of VKORC1 activity, also remains unchanged except in lungs where its expression is slightly increased to compensate for the loss of activity of VKORC1 in the lung.
The presence of these mutations in rats that can be considered laboratory rats allowed us to appreciate the consequences of a decrease in VKOR activity on the "vitamin K" status (Table 4). This status was evaluated by determining tissue vitamin K concentrations, measurement of plasma ucOC concentration, and determination of tissue calcium levels. Among the K vitamins, only MK4 was measured because the rats used received, from the age of 3 weeks up to 8 weeks, a diet supplemented with menadione as their sole source of vitamin K (the amounts of vitamin K1 in the standard feed being limited and provided by the cereals incorporated). The latter received virtually no vitamin K1 and, since there is no endogenous synthesis of vitamin K1, vitamin K1 was almost undetectable in rat tissues. Osteocalcin is a vitamin K-dependent protein produced mostly by osteoblasts [35]. In the absence of vitamin K, the concentration of ucOC increases rapidly in plasma [36] and is, therefore, considered a good marker of overall vitamin K status. Tissue calcium concentrations have also been shown to be negatively correlated with the vitamin K status. In the absence of vitamin K, MGP, which is another vitamin K-dependent protein, cannot be activated and, therefore, cannot inhibit tissue calcification [23,37,38].  The modification of the vitamin K status in the mutated rat strains does not appear to have apparent clinical consequences when animals receive a diet supplemented with menadione. In fact, the rats do not show any changes in coagulation (the quick times are comparable between strains) and do not seem to have calcification problems (tissue calcium concentrations are also comparable between strains). On the other hand, it has been shown that rats homozygous for the Y139C mutation receiving a synthetic diet (cereals replaced by starch) not supplemented with menadione for three months presented very strong vascular calcifications in testes, lungs, and kidneys [23]. This is why we evaluated the ability of different rat strains to resist a limited vitamin K intake by feeding them a Nutrients 2019, 11, 2076 13 of 16 synthetic diet without menadione (diet-K3) for a period of 12 days. To evaluate their resistance, we determined tissue MK4 concentrations (except those in liver because of the extra low concentrations), plasma ucOC concentrations, and prothrombin time, throughout the period of limited intake of vitamin K. Menadione deficiency results very rapidly into lower tissue levels of MK4 in all tissues, regardless of strains. After only four days of menadione deficiency, tissue MK4 concentrations are no longer different between strains (except in testis of wild type rats). In the absence of menadione intake, synthesis of MK4 by the enzyme UBIA1 is stopped [42,43] and tissue MK4 levels decrease to reach the same concentration in all rats. The residual quantities are potentially synthesized by the intestinal microflora [44], with the animals being all housed under the same breeding conditions, in the same rooms, and receiving all the same food. It was discovered that the presence of MK4 in the testis of wild type rats persisted longer. This persistence remains unexplained. The rapid decrease in testis of mutated strains might be due to the needs of vitamin K in other tissues to maintain the pool of activated VKDP.
This rapid decrease in MK4 concentrations is associated with a rapid increase in ucOC concentration in all mutated rats. This increase is even more rapid in the homozygous rats for the L120Q mutation with a concentration of ucOC becoming similar to that of rats homozygous for mutations Y139C and Y139F from the fourth day after the beginning of the deficiency, whereas this concentration was initially lower than that of rats homozygous for mutations Y139C and Y139F. The ucOC concentration remains unchanged in wild type rats. The same findings had already been reported for MGP in the Y139C rat with an increase in tissue ucMGP concentrations [23]. These evolutions are completely consistent with the capacity of each strain to recycle vitamin K. Since dietary vitamin K intake is limited, the gamma-carboxylation of vitamin K dependent proteins is dependent on the recycling of residual concentrations of vitamin K. The wild type rat has optimal recycling and is virtually independent of vitamin K dietary intakes. Mutated rats with limited recycling have high dietary vitamin K requirements and, when vitamin K intake is reduced, activation of vitamin K-dependent proteins becomes insufficient due to a lack of vitamin K quinone. Nevertheless, activation of hepatic vitamin K-dependent proteins (i.e., coagulation factor II, VII IX, and X) appears to be relatively preserved when compared to those of extrahepatic vitamin K dependent proteins (i.e., OC). The prothrombin times remain unchanged throughout the 12 days of deficiency, (with the exception of those of rats homozygous for the L120Q mutation which increase after 8 days of deficiency), whereas the ucOC concentration increases from the fourth day of deficiency. Since the liver is the first organ to capture dietary vitamin K, it is possible that vitamin K1 present in reduced amounts in food may maintain activated clotting factor concentrations at levels consistent with a normal prothrombin time [45]. In rats homozygous for the L120Q mutation, the recycling efficiency being the lowest (3 and 1.5 times lower than the homozygous rats for the Y139F and Y139C mutation, respectively), the low dietary intake is no longer sufficient to maintain the pools of activated clotting factors at sufficient levels.
In this study, we clearly showed that coding mutations of the Vkorc1 gene could affect the vitamin K status due to failing vitamin K recycling. This change in the vitamin K status has no apparent clinical consequences if vitamin K dietary intake is sufficient. In case of insufficient dietary vitamin K intake, these mutations can lead to insufficient activation of extrahepatic vitamin K-dependent proteins that can lead to vascular calcification due to the absence of MGP activation [23,46] changes in glucose homeostasis due to the increase in plasma ucOC described as an osteoblast-derived hormone [47], or even coagulation problems in the case of mutations severely affecting the efficiency of vitamin K recycling. Nevertheless, during this study, only three mutations were characterized. The three mutated amino acids are all localized in the fourth transmembrane domain containing the catalytic site [31], whereas, in humans, mutations have been detected in the four transmembrane domains and in the cytosolic loop and do not always seem to affect VKORC1 activity [30]. Further studies will be needed to evaluate the impact of each mutation on the vitamin K status. Nevertheless, the prevalence of these mutations in patients with vascular calcifications could be informative with regard to their impact on vitamin K status.