K-deficient Rats Research Articles

Tissue Distribution of Menaquinone-7 and the Effect of α-Tocopherol Intake on Menaquinone-7 Concentration in Rats.

We have reported that vitamin E intake lowers phylloquinone (PK) concentration in extrahepatic tissues of rats. In this study, we aimed to clarify the characteristic of the distribution of menaquinone-7 (MK-7), a vitamin K contained in fermented foods, by comparison with other vitamin K distributions and to clarify the effect of vitamin E intake on MK-7 concentration in rats. Rats were fed a vitamin K-free diet (Free group), a diet containing 0.75 mg PK/kg (PK group), a 0.74 mg menaquinone-4 (MK-4)/kg diet (MK-4 group), a 1.08 mg MK-7/kg diet (MK-7 group), or a 0.29 mg menadione (MD)/kg diet (MD group) for 16 wk. MK-7 mainly accumulated in the liver, spleen, and adrenal gland of the MK-7 group, although PK accumulated in the serum and all tissues of the PK group. Conversely, MK-4 was present in all tissues of the PK, MK-4, MK-7, and MD groups. MK-4 concentration in the serum, liver, adipose tissue, and spleen was higher in the MK-4 group than in the other groups; however, MK-4 concentration in the kidney, testis, tibia, and brain was lower in the MK-4 group than in the PK, MK-7, and MD groups. Next, vitamin E- and K-deficient rats were orally administered MK-7 with or without α-tocopherol. α-Tocopherol did not affect MK-7 or MK-4 concentration in the serum and various tissues. These results suggested that MK-7 is particularly liable to accumulate in the liver, and MK-7 concentration is not affected by vitamin E intake.

Differential regulation of the renal sodium-phosphate cotransporters NaPi-IIa, NaPi-IIc, and PiT-2 in dietary potassium deficiency

Dietary potassium (K) deficiency is accompanied by phosphaturia and decreased renal brush border membrane (BBM) vesicle sodium (Na)-dependent phosphate (Pi) transport activity. Our laboratory previously showed that K deficiency in rats leads to increased abundance in the proximal tubule BBM of the apical Na-Pi cotransporter NaPi-IIa, but that the activity, diffusion, and clustering of NaPi-IIa could be modulated by the altered lipid composition of the K-deficient BBM (Zajicek HK, Wang H, Puttaparthi K, Halaihel N, Markovich D, Shayman J, Beliveau R, Wilson P, Rogers T, Levi M. Kidney Int 60: 694–704, 2001; Inoue M, Digman MA, Cheng M, Breusegem SY, Halaihel N, Sorribas V, Mantulin WW, Gratton E, Barry NP, Levi M. J Biol Chem 279: 49160–49171, 2004). Here we investigated the role of the renal Na-Pi cotransporters NaPi-IIc and PiT-2 in K deficiency. Using Western blotting, immunofluorescence, and quantitative real-time PCR, we found that, in rats and in mice, K deficiency is associated with a dramatic decrease in the NaPi-IIc protein abundance in proximal tubular BBM and in NaPi-IIc mRNA. In addition, we documented the presence of a third Na-coupled Pi transporter in the renal BBM, PiT-2, whose abundance is also decreased by dietary K deficiency in rats and in mice. Finally, electron microscopy showed subcellular redistribution of NaPi-IIc in K deficiency: in control rats, NaPi-IIc immunolabel was primarily in BBM microvilli, whereas, in K-deficient rats, NaPi-IIc BBM label was reduced, and immunolabel was prevalent in cytoplasmic vesicles. In summary, our results demonstrate that decreases in BBM abundance of the phosphate transporter NaPi-IIc and also PiT-2 might contribute to the phosphaturia of dietary K deficiency, and that the three renal BBM phosphate transporters characterized so far can be differentially regulated by dietary perturbations.

Difference in the metabolism of vitamin K between liver and bone in vitamin K-deficient rats

The difference between vitamin K metabolism in the liver and that in the bone of vitamin K-deficient rats was examined. After 17 d administration of vitamin K-deficient food, vitamin K in the liver was almost depleted, and prothrombin time (PT) was prolonged. Serum total osteocalcin level was slightly decreased by vitamin K deficiency, whereas serum undercarboxylated osteocalcin level did not change. The level of menaquinone (MK)-4 as well as that of phylloquinone was decreased, but approximately 40 % of the initial level still existed in the femur after the 17 d period. A single-dose administration of vitamin K (250 nmol/kg body weight) markedly increased vitamin K level in the liver but not in the femur. These results suggest that the turnover of vitamin K in the bone is slower than that in the liver, and bone metabolism may be little affected by the short period of intake of vitamin K-deficient food. However, intake of a larger amount of vitamin K is required for its accumulation in the bone than in the liver. Furthermore, the counteracting effect of MK-7 on prolonged PT in vitamin K-deficient rats was found to be higher than phylloquinone or MK-4.

Glycosphingolipids modulate renal phosphate transport in potassium deficiency

Potassium (K) deficiency (KD) and/or hypokalemia have been associated with disturbances of phosphate metabolism. The purpose of the present study was to determine the cellular mechanisms that mediate the impairment of renal proximal tubular Na/Pi cotransport in a model of K deficiency in the rat. K deficiency in the rat was achieved by feeding rats a K-deficient diet for seven days, which resulted in a marked decrease in serum and tissue K content. K deficiency resulted in a marked increase in urinary Pi excretion and a decrease in the V(max) of brush-border membrane (BBM) Na/Pi cotransport activity (1943 +/- 95 in control vs. 1184 +/- 99 pmol/5 sec/mg BBM protein in K deficiency, P < 0.02). Surprisingly, the decrease in Na/Pi cotransport activity was associated with increases in the abundance of type I (NaPi-1), and type II (NaPi-2) and type III (Glvr-1) Na/Pi protein. The decrease in Na/Pi transport was associated with significant alterations in BBM lipid composition, including increases in sphingomyelin, glucosylceramide, and ganglioside GM3 content and a decrease in BBM lipid fluidity. Inhibition of glucosylceramide synthesis resulted in increases in BBM Na/Pi cotransport activity in control and K-deficient rats. The resultant Na/Pi cotransport activity in K-deficient rats was the same as in control rats (1148 +/- 52 in control + PDMP vs. 1152 +/- 61 pmol/5 sec/mg BBM protein in K deficiency + PDMP). These changes in transport activity occurred independent of further changes in BBM NaPi-2 protein or renal cortical NaPi-2 mRNA abundance. K deficiency in the rat causes inhibition of renal Na/Pi cotransport activity by post-translational mechanisms that are mediated in part through alterations in glucosylceramide content and membrane lipid dynamics.

Acetazolamide in Hypokalemic Periodic Paralysis

The mechanism of action of acetazolamide in the K-deficient diet rat, an animal model of human hypokalemic periodic paralysis (hypoPP), was investigated at the University of Bari, Italy.

Intestinal, hepatic, and circulating vitamin K levels at low and high intakes of vitamin K in rats

The aim of the present study was to assess how high doses of dietary vitamin K influence the intestinal profile of K-vitamins in vitamin K-deficient rats, and whether the induced changes are reflected in the hepatic vitamin K store. Vitamin K-deficient rats were fed for 10 d on diets containing different forms of vitamin K, and it was determined how these diets affected the vitamin K concentration at various sites of the instestine, serum, and the liver. It was found that the absorption of phylloquinone from standard food is not more than 10%, while the absorption of pharmacological doses of oil-solubilized phylloquinone and menaquinone-4 was also far from complete (18 and 55% respectively). High intakes of phylloquinone suppress the colonic production of all higher menaquinones. High menaquinone-4 intake induces very high menaquinone-8 concentrations, both in the colonic contents as well as in the liver. These data suggest that menaquinone-4 may be converted into menaquinone-8 (but not into other menaquinones) via a metabolic pathway which has not been reported previously.

Chronic K depletion inhibits renal brush border membrane Na/sulfate cotransport

The purpose of this study was to determine if dietary potassium (K) deficiency regulates renal proximal tubular sodium gradient-dependent sulfate transport (Na/Si cotransport) in the rat and, furthermore, determine if the regulation takes place at the level of the recently cloned Na/Si cotransport system (NaSi-1). Methods and Results. Rats treated chronically (seven days) with a K-deficient diet had a significant decrease in serum Si levels and an increase in fractional excretion of ultrafilterable Si, which paralleled a significant decrease in brush border membrane (BBM) Na/Si cotransport activity. The decrease in BBM Na/Si cotransport activity was associated with decreases in BBM NaSi-1 protein and renal cortical NaSi-1 mRNA abundance. In addition, in Xenopus oocytes injected with mRNA from kidney cortex slices of K-deficient rats, there was a significant reduction in the induced Na/Si cotransport, whereas there was no alteration in l-leucine uptake, suggesting that in K-deficient rats, there is a specific decrease in functional mRNA encoding the NaSi-1 mRNA. These findings indicate that chronic K deficiency leads to a reduction in serum Si levels and an increase fractional excretion of Si, and reduces Si reabsorption by down-regulating the expression of the proximal tubular Na/Si-1 cotransporter protein and mRNA.

Induction of prothrombin synthesis by K-vitamins compared in vitamin K-deficient and in brodifacoum-treated rats

Vitamin K is a group name for a number of prenylated 2-methyl-1,4-naphtoquinones, which may differ in their ability to function as a cofactor for prothrombin biosynthesis. To quantify the bioactivity of different forms of vitamin K, two experimental animal systems are frequently used: vitamin K-deficient rats and anticoagulated rats. In this paper both models are compared, and it is shown that the results obtained depend on the model used. The main reason for this discrepancy is the difference in recycling of vitamin K-epoxide, which results in a 500 times higher vitamin K requirement in anticoagulated rats. Absorption and hepatic accumulation of long chain menaquinones seem to be restricted to a maximum, whereas also the lipophilic nature of long chain menaquinones may hamper the quinone–quinol reduction in anticoagulated animals. If these data may be extrapolated to patients, food items rich in K 1 and MK-4 would be expected to influence the stability of oral anticoagulation to a much larger extent than food items primarily containing higher menaquinones.

Tissue distribution of K-vitamers under different nutritional regimens in the rat

Two forms of vitamin K [phylloquinone (K 1) and menaquinone-4 (MK-4)] were added to vitamin K-deficient rat food in varying amounts. These diets were given as the sole source of nutrition to rats for one week. The minimal dietary requirements (MDR) to attain maximal prothrombin synthesis were determined to be 0.6 and 6–10 μg/g of food for K 1 and MK-4, respectively. The difference between both vitamers could be explained by the limited hepatic accumulation of MK-4. Next, vitamin K was offered to rats at concentrations ranging between 0.6 and 3000 μg/g of food, and the tissue distribution of vitamin K was investigated after one week of administration. Accumulation of K 1 and MK-4 was found in all tissues investigated, but both the absolute tissue concentration and the ratio between K 1 and MK-4 were tissue-dependent. Highest values were found in liver and in heart, but since the heart contains no γ-glutamylcarboxylase, the function of vitamin K in this tissue remains obscure. High tissue concentrations of MK-4 were also found in pancreas and testis after a diet containing K 1 exclusively. The data indicate that this conversion is tissue-specific, but neither the reason nor its mechanism are known.

Bioavailability of phylloquinone and menaquinones after oral and colorectal administration in vitamin K-deficient rats

Rats were made vitamin K-deficient by feeding them a diet devoid of vitamin K and by rigorously preventing coprophagy. After one week, circulating prothrombin concentrations were between 5 and 10% of initial values, and various amounts of phylloquinone. menaquinone-4, and menaquinone-9 were given in a single dose either subcutaneously, orally, or colorectally. The relative ‘vitamin K activities’ of these compounds were assessed by comparing their ability to support prothrombin synthesis after subcutaneous injection. Intestinal and colonic absorption were deduced from the difference between subcutaneous and either oral or colorectal administration of the vitamers. It is concluded that the colonic absorption of all three forms of vitamin K is extremely poor, suggesting that physiological menaquinones in the colon do not contribute substantially to vitamin K status in rats. Furthermore, the stimulation of prothrombin synthesis by menaquinone-9 lasted much longer than that by the two other K-vitamers, resulting in a substantially higher ‘vitamin K activity’ of menaquinone-9.

The relative effects of phylloquinone and menaquinone-4 on the blood coagulation factor synthesis in vitamin K-deficient rats

Rats were made vitamin K-deficient by feeding them a 1:1 (w/w) mixture of a commercial vitamin K-depleted diet and boiled white rice. After one week of treatment the rats had developed severe vitamin K deficiency, resulting in Thrombotest® values of 5–10% of the initial values. In this experimental system the efficacy of phylloquinone (K 1) was compared with that of menaquinone-4 (MK-4) by measuring the extent to which the Thrombotest was normalized after the administration of varying doses of the respective vitamins. Oral administration of the vitamins showed that the efficacy of K 1 was at least two-fold higher than that of MK-4. As comparable results were obtained after subcutaneous administration of the vitamins, we conclude that after oral administration the intestinal absorption had been quick and nearly complete. A less pronounced effect of K 1 and MK-4 was found after colorectal administration. For both forms of vitamin K relatively high amounts (well above the physiological concentration) were required before significant effects on the Thrombotest could be observed. Therefore these data demonstrate the importance of sufficient dietary vitamin K consumption in rats. The efficacy of other menaquinones may be investigated in the same experimental animal model system.

Effects of potassium deficiency on growth and protein synthesis in skeletal muscle and the heart of rats.

The effects of potassium deficiency on growth, K content and protein synthesis have been compared in 4-13-week-old rats. When maintained on K-deficient fodder (1 mmol/kg) rats ceased to grow within a few days, and the incorporation of [3H]leucine into skeletal muscle protein in vivo was reduced by 28-38%. Pair-feeding experiments showed that this inhibition was not due to reduced energy intake. Following 14 d on K-deficient fodder, there was a further reduction (39-56%) in the incorporation of [3H]leucine into skeletal muscle protein, whereas the incorporation into plasma, heart and liver proteins was not affected. The accumulation of the non-metabolized amino acid alpha-aminoisobutyric acid in the heart and skeletal muscles was not reduced. The inhibitory effect of K deficiency on 3H-labelling of muscle protein was seen following intraperitoneal (10-240 min) as well as intravenous (10 min) injection of [3H]leucine. In addition, the incorporation of [3H]phenylalanine into skeletal muscle protein was reduced in K-depleted animals. Following acute K repletion in vivo leading to complete normalization of muscle K content, the incorporation of [3H]leucine into muscle protein showed no increase within 2 h, but reached 76 and 104% of the control level within 24 and 72 h respectively. This was associated with a rapid initial weight gain, but normal body-weight was not reached until after 7 weeks of K repletion. Following 7 d on K-deficient fodder the inhibition of growth and protein synthesis was closely correlated with the K content of the fodder (1-40 mmol/kg) and significant already at modest reductions in muscle K content. In vitro experiments with soleus muscle showed a linear relationship between the incorporation of [3H]leucine into muscle protein and K content, but the sensitivity to cellular K deficiency induced in vitro was much less pronounced than that induced in vivo. Thus, in soleus and extensor digitorum longus (EDL) muscles prepared from K-deficient rats, the incorporation of [3H]leucine was reduced by 30 and 47% respectively. This defect was completely restored by 24 h K repletion in vivo. It is concluded that in the intact organism protein synthesis and growth are very sensitive to dietary K deficiency and that this can only partly be accounted for by the reduction in cellular K content per se. The observations emphasize the need for adequate K supplies to ensure optimum utilization of food elements for protein synthesis and growth.

Depression of liver microsomal vitamin K epoxide reductase activity associated with antibiotic-induced coagulopathy

Hypoprothrombinemic changes in blood coagulation parameters, such as prolongation of prothrombin time, increase in the level of plasma protein induced by vitamin K absence, and decrease in plasma prothrombin level, were detected in rats fed a vitamin K-deficient diet. These changes were enhanced by the administration of β-lactam antibiotics containing N-methyltetrazolethiol, thiadiazolethiol or methyl-thiadiazolethiol. Microsomal vitamin K epoxide reductase activity was suppressed with the maximum effect at 1–2 days after the treatment and with recovery, thereafter, gradually to the normal level after 5–7 days. Hypoprothrombinemic alterations in blood coagulation parameters following a single administration of antibiotic to vitamin K-deficient rats were somewhat delayed compared with the change in the epoxide reductase activity, but the effects of the antibiotic on both blood coagulation parameters and the enzyme activity disappeared completely 7 days after the antibiotic treatment. Antibiotic-induced depression of the epoxide reductase activity was observed even in the vitamin K sufficient rats, although the hypoprothrombinemic changes in the blood coagulation parameters did not develop. Vitamin K administration could normalize the blood coagulation parameters in the hypoprothrombinemic rats caused by treatment with the antibiotics but without recovery of the decreased epoxide reductase activity. These results suggest that some antibiotics inhibit liver microsomal vitamin K epoxide reductase, which causes hypoprothrombinemia to develop under vitamin K-deficient conditions.

In vivo effects of β-lactam antibiotics and heterocyclic thiol compounds on vitamin K-dependent carboxylation activity and blood coagulation factors in vitamin K-deficient rats

The vivo effects of heterocyclic thiol compounds, corresponding to the 3′-position substituents of several β-lactam antibiotics, on blood coagulation factors and on liver microsomal γ-glutamylcarboxylation (γ-carboxylation) activity were evaluated in rats maintained on a vitamin K-deficient diet. These rats, when compared to normal control animals, exhibited hypoprothrombinemic changes: prolongation of both prothrombin time and activated partial thromboplastin time, decreases in factor VII and plasma prothrombin, and increases in PIVKA II (descarboxyprothrombin) both in plasma and liver. They also displayed a marked increase in liver microsomal γ-carboxylation activity. These blood coagulation variables could be altered markedly by administering various heterocyclic thiol compounds to the vitamin K-deficient rats, although these compounds did not inhibit γ-carboxylation activity in an assay system using phylloquinone. A similar pattern of alteration was observed when some β-lactam antibiotics were administered. Increased microsomal γ-carboxylation activity in antibiotic-treated vitamin K-deficient rats was normalized by the administration of vitamin K, concomitant with the recovery of blood coagulation variables to the normal range. The results indicate that antibiotic-induced hypoprothrombinemia in vivo is not caused by inhibition of enzymes of the γ-carboxylation system, such as vitamin K reductase and γ-glutamylcarboxylase, but is related to the endogenous vitamin K level.

Vitamin K Reversible Hypoprothrombinemia in Rats II. Efficacy of Vitamin K on Latamoxef-Induced Coagulopathy in Rats

Feeding of a vitamin K-deficient diet caused the development of hypo-prothrombinemic changes in rats such as prolongation of prothrombin time (PT) and activated partial thromboplastin time (APTT), decreases in plasma prothrombin and clotting factor VII levels, and an increase in the descarboxyprothrombin (PIVKA) level in both plasma and liver. Successive administrations of latamoxef (LMOX) to the vitamin K-deficient rats resulted in the further enhancement of these changes. After the development of hypoprothrombinemia with LMOX, a single subcutaneous injection of vitamin K1 normalized most of these abnormalities in blood coagulation parameters within 6 hr. When vitamin Κ was given at 200 μg/kg, PT, APTT and the plasma PIVKA level showed normal values for at least 8 days even when the animals were fed a vitamin K-deficient diet and treated with LMOX during the recovery period. The amount of vitamin Κ required to maintain most of the blood coagulation parameters in the normal range was about 3 μg/kg/day. The plasma level of vitamin Κ was higher than 0.3-0.5 ng/ml when the blood coagulation parameters were maintained in the normal range.

Vitamin K reversible hypoprothrombinemia in rats. II. Efficacy of vitamin K on latamoxef-induced coagulopathy in rats.

Feeding of a vitamin K-deficient diet caused the development of hypoprothrombinemic changes in rats such as prolongation of prothrombin time (PT) and activated partial thromboplastin time (APTT), decreases in plasma prothrombin and clotting factor VII levels, and an increase in the descarboxyprothrombin (PIVKA) level in both plasma and liver. Successive administrations of latamoxef (LMOX) to the vitamin K-deficient rats resulted in the further enhancement of these changes. After the development of hypoprothrombinemia with LMOX, a single subcutaneous injection of vitamin K1 normalized most of these abnormalities in blood coagulation parameters within 6 hr. When vitamin K was given at 200 micrograms/kg, PT, APTT and the plasma PIVKA level showed normal values for at least 8 days even when the animals were fed a vitamin K-deficient diet and treated with LMOX during the recovery period. The amount of vitamin K required to maintain most of the blood coagulation parameters in the normal range was about 3 micrograms/kg/day. The plasma level of vitamin K was higher than 0.3-0.5 ng/ml when the blood coagulation parameters were maintained in the normal range.

Production of hypoprothrombinaemia by cefazolin and 2-methyl-1,3,4-thiadiazole-5-thiol in the rat.

Recent findings have suggested that the 1-methyltetrazole-5-thiol (MTT) group contained in several beta-lactam antibiotics may be responsible for the hypoprothrombinaemia associated with these drugs. In order to determine if the hypoprothrombinaemia associated with cefazolin is due to the presence of the structurally related 2-methyl-1,3,4,-thiadiazole-5-thiol (MTD) group which it possesses, the ability of MTD and cefazolin to produce hypoprothrombinaemia in rats was examined. Female rats maintained on a vitamin K-deficient diet for ten days developed hypoprothrombinaemia after the intravenous administration of cefazolin for two subsequent days. Hypoprothrombinaemia was also produced by the oral administration to vitamin K-deficient rats of either cefazolin or MTD, while the oral administration of cefotaxime, which does not contain a thiol group, had no effect. In a rat liver microsomal system, MTD was found to be a much more potent inhibitor than cefazolin of the vitamin K-dependent step in clotting factor synthesis, the gamma-carboxylation of glutamic acid. These results suggest that the hypoprothrombinaemia associated with cefazolin may be due to the MTD group.

Effect of sex hormones on hypoprothrombinemia induced by N-Methyltetrazolethiol in rats

N-Methyltetrazolethiol (NMTT) increased prothrombin time (PT) and decreased plasma factor VII and prothrombin levels only in vitamin K-deficient male rats. In female rats identical treatment with NMTT did not produce hypoprothrombinemia. Conventional and germ-free rats displayed no significant difference in the manifestation of hypoprothrombinemia, but the increase of PT in NMTT-treated vitamin K-deficient rats was greater in the germ-free males. Estradiol administration or castration of male rats retarded manifestation of vitamin K deficient syndromes such as increases of PT and activated partial thromboplastin time (APTT), decreases of plasma factor VII and prothrombin levels, and increases of plasma and liver descarboxyprothrombin (PIVKA) levels, and testosterone injection to the castrated rats restored these changes. In female rats testosterone treatment or castration enhanced the manifestation of hypoprothrombinemia and estradiol treatment to the castrated females retarded it. Gamma-glutamyl-carboxylase activity was increased by vitamin K-deficiency but not inhibited by testosterone or NMTT. These data suggest that estrogen protects the rat against manifestation of hypoprothrom-binemia even with NMTT treatment, while androgen enhances vitamin K deficiency, and supplementation of vitamin K prevents its deficiency in NMTT-treated rats.

Vitamin K-dependent carboxylase.

Vitamin K-dependent carboxylase.

Behavioral perturbations in the vitamin K-deficient rat

Anecdotal observations of the behavior of rats with a vitamin K-deficiency suggested that this deficiency was associated with hypoactivity, general malaise, and a lack of exploratory behavior. These observations were pursued by assessing locomotor activity in a circular photocell-monitored track, open-field activity, and radial-arm maze performance in rats rendered vitamin K-deficient by dietary depletion or by warfarin treatment. There was a significant reduction (approximately 25% at the median) in the locomotor activity of dietary vitamin K-deficient rats compared with rats fed a control diet. In the open-field, warfarin administration was associated with a significant shift from more exploratory behaviors to less exploratory behaviors. Consistent with these findings, radial-arm maze assessment showed a comparative reduction in locomotor activity in the dietary vitamin K-deficient rats with no alteration in performance, i.e., short-term memory. These animal behavioral studies suggest that sub-clinical and clinical vitamin K-deficiency may contribute to physical and psychiatric symptomatology.