Research article Received: 18 November 2014,
Revised: 30 March 2015,
Accepted: 2 May 2015
Published online in Wiley Online Library: 2 June 2015
(wileyonlinelibrary.com) DOI 10.1002/bmc.3506
Vitamin K1 distribution following intravenous vitamin K1–fat emulsion administration in rats Xue Xiaoa, Yan-Ni Mia, Fa Wangb, Bing-Hua Zhangb, Lei Caoa* and Yong-Xiao Caoa ABSTRACT: This study investigated vitamin K1 (VK1) distribution following intravenous vitamin K1–fat emulsion (VK1–FE) administration and compared it with that after VK1 injection. Rats were intravenously injected with VK1–FE or VK1. The organ and tissue VK1 concentrations were determined using high-performance liquid chromatography method at 0.5, 2 and 4 h to determine distribution, equilibrium and elimination phases, respectively. In the VK1–FE group, the plasma, heart and spleen VK1 concentrations decreased over time. However, other organs like liver, lung, kidney, muscle and testis, reached peak VK1 concentrations at 2 h. In the VK1 injection group, the liver VK1 concentrations were significantly higher than those in other organs at the three time points. However, VK1 concentrations in the other organs peaked at 2 h. In addition, in VK1–FE group, the heart, spleen and lung VK1 concentrations were significantly higher than those in the VK1 injection group at the three time points, and the liver VK1 concentration was significantly higher than that in the VK1 injection group at 4 h. The VK1 amount was greatest in the liver compared with the other organs. Thus, the liver is the primary organ for VK1 distribution. The distribution of VK1 is more rapid when injected as VK1–FE than as VK1. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: vitamin K1; vitamin K1-fat emulsion; distribution; high-performance liquid chromatography
Introduction Vitamin K1 (VK1) is essential for the formation of prothrombin and coagulation factors II, VII, IX and X. These factors are glycoproteins with a number of γ-carboxyglutamic acid residues clustered at the N-terminal end of the peptide chain (Holmes et al., 2012). VK1 also acts on proteins C and S, as well as non-coagulation proteins, such as osteocalcin and matrix Gla protein, which are involved in the metabolic pathways of bone and other tissues (Marinova et al., 2013). VK1 is mainly used to treat and/or prevent VK1 deficiency, specifically in newborns (Plank et al., 2013), and to antagonize the effects of warfarin (Holmes et al., 2012). Recently, the number of severe adverse reactions caused by the intravenous administration of VK1 has increased and attracted public attention (Fiore et al., 2001; Riegert-Johnson et al., 2001). Because VK1 is a lipid-soluble substance and is poorly soluble in water, a solubilizer, such as Tween 80, is required to prepare it for injection (Qiu et al., 2013). However, Tween 80 as a drug accessory has recently been implicated in severe adverse clinical effects. To avoid using a solubilizer, a new vitamin K1–fat emulsion (VK1–FE) preparation has been developed (Mi et al., 2014). We speculated that VK1–FE could change the distribution characteristics of VK1 and that the VK1 concentrations in some tissues of intravenously VK1–FE-injected animals could be changed. The objective of the present study was to investigate the VK1 distribution after intravenous VK1–FE administration and to compare it with that after VK1 injection to illustrate the VK1 distribution characteristics of these two preparations in rats.
Materials and methods
(Xi’an, China), respectively. VK1 standard was purchased from the National Institute for Food and Drug Control (Beijing, China), and its purity was >98% (high-performance liquid chromatography, HPLC). The injectable heparin sodium was purchased from Xingmao Co. (Shanghai, China). Ethanol and cyclohexane were obtained from Kemiou Reagent Co. (Tianjin, China) and were of HPLC grade.
Animals and drug administration Sprague–Dawley rats weighing 200–220 g and Kunming mice weighing 18–22 g (half males and half females) were purchased from the Animal Center of Xi’an Jiaotong University (Xi’an, China). The animals were maintained in conventional laboratory conditions for temperature, humidity and light and allowed food (standard pellet diet) and tap water ad libitum. The study was approved by the Ethics Committee at Xi’an Jiaotong University College of Medicine. Sixty mice were randomly divided into 10 groups, and a single dose of 2 mg/kg VK1–FE was injected into mice via the tail vein. The livers were collected at 0, 0.17, 0.5, 1, 2, 4, 6, 8, 10 and 12 h after the injection (Choonara et al., 1985). The rats were fasted for 24 h, but had free access to water. Then, the rats were randomly divided * Correspondence to: Lei Cao, Department of Pharmacology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi, 710061, China. Email: [email protected] a
Department of Pharmacology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi, 710061, China
b
Shaanxi Institute for Food and Drug Control, Xi’an, Shaanxi, 710061, China
Drugs and reagents
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Abbreviations used: VK1, vitamin K1; VK1–FE, vitamin K1–fat emulsion.
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The injectable VK1–FE and VK1 were purchased from Cisen Pharmaceutical Co. (Shandong, China) and Anjian Pharmaceutical Co.
X. Xiao et al. into three groups: the control group (n = 8); the VK1–FE group (n = 27); and the VK1 injection group (n = 27). Rats from the latter two groups were injected with a single dose (1 mg/kg) of VK1–FE or VK1 via the small saphenous vein. The blood, organ and tissue samples, such as the heart, liver, spleen, lung, kidneys, brain, testis, muscle and fat, were collected at 0.5 and 4 h after injection (de Boer et al., 2005). Instruments and chromatographic conditions Chromatographic analyses were performed under ambient conditions with a Waters Alliance HT HPLC system (Waters Association, Milford, MA). This system is a ZMPOWER-2 chromatography workstation. It has a variable wavelength programmable UV spectrophotometric detector (Waters 2489), which was operated at 254 nm, and an automated injection system. Chromatographic separations were achieved using a Kromasil 100-5 C18 column (150 × 4.6 mm, o.d., 8 mm, Akzonobel, Sweden). The mobile phase consisted of ethanol–water–diethyl ether (88:2:10, v/v) at a flow rate of 1.5 mL/min. The column oven temperature was 30 °C, and the injection volume was 50 μL. Blood and tissue sample preparation Blood samples were collected from the abdominal aorta, transferred into a 5 mL lithium heparin anticoagulant tube and immediately protected from light. Then, plasma was obtained by centrifugation (2500 rpm/10 min). One milliliter of plasma was deproteinized with 3 mL absolute ethyl alcohol, and then 3 mL cyclohexane was added and mixed vigorously. The mixture was centrifuged (12,000 rpm/5 min), and then the cyclohexane was removed and evaporated. To obtain the residue, 1 mL absolute ethyl alcohol was added, mixed vigorously and centrifuged twice for 5 min at 12,000 rpm, and 50 μL of the supernatant was injected into the HPLC column. The whole heart, pancreas, kidneys, lungs, testis, brain and 2.0 g liver, 2.0 g thigh muscle and 1.0 g fat from around the kidney were removed from the rats. The collected organs and tissues were cleaned, dried with filter paper and then weighed. Each organ or tissue was added to 3 mL 75% ethanol and homogenized using a Polytron homogenizer. Each homogenate was added to 3 mL cyclohexane and centrifuged. The solvent layers were isolated and dried. The resultant pellet was dissolved in 1 mL absolute ethyl alcohol, mixed vigorously, centrifuged twice for 5 min at 12,000 rpm, and then analyzed by HPLC. All of the experimental processes were performed in the dark (Qiu et al., 2013). Assay method validation To evaluate the performance of the developed extraction method, analytical parameters, including linearity, limit of detection (LOD), limit of quantification (LOQ), reproducibility, stability, recovery and precision were investigated. Protein precipitation. To develop a suitable and efficient method for VK1 extraction from rat liver, we used absolute alcohol and physiological saline for protein precipitation. Liver samples containing significantly less VKl were divided into four groups (four samples per group). The ratios of absolute alcohol to physiological saline were 4:1, 3:1, 2:1 and 1:1 (v/v). A chromatogram was recorded (Haroon and Hauschka, 1983).
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Linearity and LOD and LOQ. Blank blood and tissue samples were prepared by homogenizing fresh blood, heart, liver, spleen,
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lung, kidney, testis, muscle or fat in 75% ethanol. Linearity was evaluated within the concentration ranges of 0–100 μg/mL VK1 spiked into blank plasma and tissues. The LOD is considered the concentration that has a signal-to-noise ratio (S/N) of 3:1. The LOQ is the lowest concentration that has an S/N ratio of at least 10:1. Reproducibility. Reproducibility is defined as the variation that occurs when different analysts measure the same samples with the same procedure in different facilities. We used liver as a representative sample. Three identical samples, containing a specific amount of VK1 (1 μg/mL), were analyzed in separate HPLC runs, using the same procedure in all three runs but operated by different analysts, and the peak area of VK1 was detected. Reproducibility was determined as percentage relative standard deviation (RSD). Stability. To ensure the reliability of the results in relation to the handling and storing of plasma and tissue samples, stability studies were performed. Liver samples containing 1.0 μg/mL VK1 were prepared, and the VK1 concentrations were measured at 0, 2, 4, 6, 8, 10 and 12 h. Recovery. The recovery of VK1 from plasma and other tissues was determined at concentrations of 0.4, 2 and 10 μg/mL. Three replicates of each concentration were injected into the HPLC system. The extraction recovery at each concentration was calculated using the following equation: Recovery ð%Þ ¼ ða=bÞ100% where a is the peak area obtained from the spiked samples and b is the quantity of the spiked VK1 added. Precision. The precision of this method was assessed by adding a quantity of VK1 (2 μg/mL) to liver samples and measuring VK1 concentration using the assay procedure. Seven standard samples were prepared and assayed separately. The relationship between VK1 concentration and time in mouse liver We speculated that the liver is a major target organ of VK1 distribution. To determine the time points of VK1 distribution, the body processing of VK1 was predicted by measuring the VK1 concentration vs time curves in mouse livers. The mice were randomly divided into 10 groups (six mice per group). A single dose of 2 mg/kg VK1–FE was injected into mice via the tail vein. After 0, 0.17, 0.5, 1, 2, 4, 6, 8, 10 and 12 h, the mice were sacrificed by CO2 asphyxiation. In a dark room, the livers were removed and cleared with 0.9% (w/v) sodium chloride. The livers were dried, weighed, transferred into tubes with 1 mL 0.9% sodium chloride and 3 mL absolute alcohol, and then homogenized for 3 min using a blender. Three milliliters of cyclohexane were added and mixed vigorously. Then, the mixture was centrifuged. The cyclohexane layer was removed and evaporated (Sato et al., 2003). The residues were stored in the dark until analyzed. Tissue distribution of VK1 After 24 h of fasting, a single dose of 1 mg/kg VK1–FE or VK1 was intravenously injected into the rats. Before (n = 8) and 0.5, 2 and 4 h (n = 9) after drug injection, surgeries were performed under chloral hydrate anesthesia. A blood sample was collected from the abdominal aorta and transferred into a 5 mL lithium heparin
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Vitamin K1 distribution in rats anticoagulant tube. Plasma was obtained after centrifugation (2500 rpm/10 min) (Ducros et al., 2010). Then, the heart, liver, spleen, lungs, kidneys, brain, testis, muscle and fat were removed, cleared, dried and weighed. The tissue sample preparation procedure was the same as detailed in ’Blood and tissue sample preparation’.
Therefore, absolute alcohol–physiological saline at a 3:1 ratio was chosen as the extraction solvent for the VK1 assay. Linearity, LOD and LOQ. Under the chromatographic conditions used in this experiment, the retention time of VK1 was approximately 2.93 min (Fig. 1). The VK1 regression models in organs or tissues were represented by the following equation: y ¼ ax þ b
Data analysis Linear regression calculations were performed using Microsoft Excel 2010. The statistical analyses were performed using GRAPHPAD PRISM 5.0 (GraphPad Software, La Jolla, CA, USA). The results are presented as the mean ± SEM, and p < 0.05 was considered statistically significant. Student’s t-test was used to compare the VK1 concentration between the VK1–FE and VK1 injection groups.
The x-axis represents the VK1 concentration, and the y-axis represents the VK1 peak area. The correlation coefficient (r 2) indicates sufficient linearity. The r2 values in the nine organs and tissues ranged from 0.9980 to 1.0000. The LOQ, 10 times the S/N ratio, was 32 ng/mL. The LOD, 3 times the S/N ratio, was 14 ng/mL (Chakravarthy et al., 2015). Reproducibility. Three VK1 standard samples were analyzed in separate HPLC runs using the same procedure in all three runs and operated by three different analysts, which resulted in an RSD for the VK1 peak area of 1.682%. This result indicated that this method was consistent with the experimental requirements.
Results Assay method validation Protein precipitation. In pilot experiments, absolute alcohol, acetonitrile and methanol were selected for protein precipitation. We found that absolute alcohol produced the best results. Our purpose was to determine the optimal ratio of absolute alcohol and physiological saline for protein precipitation. The results demonstrated that the highest VK1 concentration was 4.12 ± 0.53 μg/g when the ratio was 3:1, which was considered to be the most suitable and efficient method for VK1 extraction.
Stability. Liver samples containing 1.0 μg/mL VK1 were prepared and assayed. The variation was calculated by the following equation: Variation ð%Þ ¼ ½ðx y Þ = y 100% where x is the peak area at the time point measured and y is the peak area at 0 h. Table 1 shows that the variation ranged from 8.5 to 2.4%.
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Figure 1. HPLC chromatograms of vitamin K1 (VK1). Representative chromatograms of tissues or organs at 2 h after intravenous vitamin K1–fat emulsion (VK1–FE) administration. Each chromatogram includes VK1 standard solution (A), plasma (B), heart (C), liver (D), spleen (E), lung (F), kidney (G), muscle (H), fat (I) and testis ( J).
X. Xiao et al. Table 1. vitamin K1 (VK1) stability in rat livers Detected time (h)
Peak area (μV s)
Variation (%)
41,518 41,827 41,339 42,325 42,516 41,200 41,165
— 0.74 0.43 1.94 2.4 –0.76 –0.85
0 2 4 6 8 10 12
Recovery and precision. The recovery of VK1 from different organs or tissues was determined at concentrations of 0.4, 2 and 10 μg/mL. The results are shown in Table 2. The coefficient of variation for the three concentration samples ranged from 62.76 to 102.11%. The RSD ranged from 0.03 to 2.65%. The precision of this method was assessed by adding VK1 (2 μg/mL) to seven liver samples. VK1 concentrations were determined by the assay procedure, and the RSD was 0.57%. VK1 distribution in mouse liver VK1 concentration–time curve in mouse livers. We hypothesized that the liver is the major target organ of VK1 distribution. To determine the time points of VK1 distribution, VK1 present in the body was predicted by measuring its concentrations in mouse livers. Curves of VK1 concentration vs time were then constructed. The results are shown in Fig. 2. The VK1 concentration peaked (maximum of 11.00 ± 1.44 μg/g) at 2 h after VK1–FE intravenous administration, and then it consistently declined. At 12 h, the concentration was close to the level at 0 h. From the VK1 concentration–time curve, we selected 0.5, 2 and 4 h to represent the distribution, equilibrium and elimination phases of VK1, respectively. VK1 distribution in rats There were no significant differences in body weight or organ weights in the rats between the VK1 injection group and the VK1–FE group at the three time points (Table 3). After 24 h of fasting, VK1 was not detected in any of the organs or tissues. Distribution phase. Thirty minutes after administration was selected to represent the distribution phase of VK1. VK1 distribution Table 2. VK1 recovery in the plasma and other tissues or organs (n = 3–5, mean ± SEM) The recovery of different VK1 concentrations (%)
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Plasma Heart Liver Spleen Lung Kidney Muscle Fat Testis
0.4 μg/mL
2 μg/mL
10 μg/mL
99.34 ± 0.54 82.72 ± 0.44 88.61 ± 1.12 67.27 ± 1.10 92.09 ± 0.55 89.63 ± 0.34 84.81 ± 0.28 67.16 ± 1.53 89.70 ± 1.56
102.11 ± 0.60 81.32 ± 0.74 84.24 ± 0.16 63.27 ± 0.02 91.89 ± 0.02 85.74 ± 0.08 86.85 ± 0.85 64.99 ± 0.05 92.03 ± 0.07
100.58 ± 0.46 83.00 ± 0.02 88.13 ± 0.19 70.15 ± 0.41 91.88 ± 0.10 86.16 ± 0.98 88.40 ± 0.47 62.76 ± 0.07 93.81 ± 1.60
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Figure 2. Liver VK1 amount–time profile following the injection of a single dose of VK1–FE in mice. The data are given as the mean ± SEM, n = 8.
in various organs and tissues were determined. First, VK1 concentrations (μg/g) were measured in different organs and tissues. Then, the amounts of VK1 (μg) in the various organs were calculated according to their weights. The results showed that, in the VK1 injection group, the concentration and amount of VK1 in the liver were higher than in the other organs, including the heart, spleen, lung, kidney, muscle, fat, testis and blood. In the VK1–FE group, the concentration and/or amount of VK1 in the heart, liver, and spleen were higher than those in the other organs and tissues, such as the lung, kidney, muscle, fat and testis. The concentrations and amounts of VK1 in the heart, spleen, and lung in the VK1–FE group were significantly higher than those of the VK1 injection group (p < 0.01). However, the concentrations and the total amount of VK1 in the liver, kidney, muscle, fat, and testis were not significantly different between the two preparations (p > 0.05; Fig. 3). Equilibrium phase. Two hours after drug administration was chosen to represent the equilibrium phase. In the VK1 injection group, the concentration and amount of VK1 in liver were higher than those in other organs and tissues, such as the blood, heart, spleen, lung, kidney, muscle, fat and testis. In the VK1–FE group, the concentrations and/or amounts of VK1 were higher in the heart, liver and spleen than those in the other organs and tissues, such as the lung, kidney, muscle, fat, testis and plasma. However, it was not detected in the brain in either the VK1–FE or the VK1 injection group. The VK1 concentrations and amounts in the heart, spleen and lung in the VK1–FE group were significantly higher than those in the VK1 injection group (p < 0.01), while the VK1 concentration in plasma was significantly lower than in the VK1 injection group (p < 0.05). There was no significant difference in the VK1 concentrations and/or the amounts in the liver, spleen, muscle and testis between the two preparations (p > 0.05; Fig. 4). Elimination phase. Four hours after drug administration was selected to represent the elimination phase. The results showed that the VK1 concentrations in the plasma, heart, liver, spleen, lung, kidney and muscle at 4 h were lower than those at 2 h in both the VK1 injection group and VK1–FE group. The
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Vitamin K1 distribution in rats Table 3. Rat body and organ weights in the VK1 and VK1–FE injection groups (n = 4–9, mean ± SEM) Weight at 30 min ( g)
Weight Heart Liver Spleen Lung Kidney Testis
Weight at 2 h ( g)
Weight at 4 h ( g)
VK1–FE
VK1
VK1–FE
VK1
VK1–FE
VK1
200 ± 2.7 0.66 ± 0.02 7.20 ± 0.17 0.52 ± 0.04 1.11 ± 0.02 2.06 ± 0.01 2.27 ± 0.11
198 ± 4.3 0.67 ± 0.02 7.10 ± 0.17 0.53 ± 0.02 1.03 ± 0.02 1.89 ± 0.05 2.25 ± 0.05
199 ± 2.7 0.67 ± 0.02 7.88 ± 0.15 0.50 ± 0.03 1.13 ± 0.03 1.93 ± 0.08 2.11 ± 0.16
198 ± 1.7 0.67 ± 0.03 8.28 ± 0.22 0.50 ± 0.03 1.12 ± 0.03 1.88 ± 0.04 2.27 ± 0.16
206 ± 2 0.71 ± 0.01 9.12 ± 0.23 0.61 ± 0.22 1.17 ± 0.06 1.83 ± 0.03 1.93 ± 0.05
204 ± 2 0.68 ± 0.02 8.81 ± 0.51 0.57 ± 0.03 1.10 ± 0.03 1.73 ± 0.04 1.91 ± 0.11
Figure 3. The concentrations (A) and amounts (B) of VK1 in the organs at 30 min after 1 mg/kg intravenous administration of VK1–FE or VK1 in rats. The data are given as the means ± SEM. Heart and lung, n = 9; liver and spleen, n = 8; kidney, n = 6; testis, n = 4. **p < 0.01 vs the VK1 injection group.
Figure 4. The concentrations (A) and amounts (B) of VK1 in the organs at 2 h after 1 mg/kg intravenous administration of VK1–FE or VK1. The data are given as the mean ± SEM. Blood and lung, n = 9; heart, liver and spleen, n = 8; kidney, n = 6; testis, n = 4. **p < 0.01 vs the VK1 injection group.
liver VK1 concentration decreased rapidly in the VK1 injection group. The concentrations and amounts of VK1 in the heart, spleen, lung and liver remained significantly higher in the VK1–FE group than in the VK1 injection group (p < 0.01). There was no significant difference in the VK1 concentrations and amounts in the kidney, muscle, fat and testis between the VK1–FE and VK1 injection groups (p > 0.05; Fig. 5). Distribution changes of VK1 in various organs and tissues over time
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Liver. The liver VK1 concentration was not the highest among the organs in the VK1–FE group (Figs. 3A, 4A and 5A). Because
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Plasma. Before the intravenous administration of VK1, it was not detected in the plasma. In the VK1 injection group, the plasma VK1
concentrations at 0.5, 2 and 4 h were 0.92 ± 0.20, 0.26 ± 0.03 and 0.04 ± 0.01 μg/mL, respectively, indicating a gradual decrease in the concentration. Thirty minutes after VK1–FE administration, the VK1 concentration was 0.45 ± 0.15 μg/mL, which was approximately half of the concentration in the VK1 injection group at this time point. The VK1 concentrations were 0.11 ± 0.04 and 0.06 ± 0.01 μg/mL at 2 and 4 h, respectively, in the VK1–FE group. At 2 h, the plasma VK1 concentration in the VK1 injection group was 2.3-fold higher than that in the VK1–FE group. Although the plasma VK1 concentration decreased over time, the reduction was more rapid in the VK1 injection group than in the VK1–FE group.
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Figure 5. The concentrations (A) and amounts (B) of VK1 in the organs at 4 h after 1 mg/kg intravenous administration of VK1–FE or VK1. The data are given as the mean ± SEM. Blood, heart, spleen and lung, n = 9; liver, n = 8; kidney, n = 6; testis, n = 4. **p < 0.01 vs the VK1 injection group.
the mass of the liver is large, the amount of VK1 in the liver was the highest amongst the organs (Figs. 3B, 4B and 5B), suggesting that the liver is the most important organ affecting VK1 distribution. Figure 6 shows the changes in VK1 concentrations and amounts in the liver over time. Thirty minutes after the intravenous injection of VK1–FE or VK1, the VK1 concentrations in the livers were at the same level, 2.27 ± 0.39 or 2.32 ± 0.28 μg/g, respectively (p > 0.05). At 2 h, the VK1 concentrations in the VK1–FE and VK1 injection groups were 3.60 ± 0.69 and 3.34 ± 0.46 μg/g, respectively (p > 0.05), which were higher than those at 30 min. The VK1 concentration was significantly higher in the VK1–FE group than in the VK1 injection group at 4 h (p 0.05). Brain and testis. The testis VK1 concentrations and amounts at the three time points were very low in the two preparations and had no impact on its distribution. VK1 was not detected in the brain at the three time points.
Discussion An emulsion carrier can increase the water solubility of a poorly soluble compound, changing the distribution of the drug in the body, increasing the drug permeability of biological membranes and affecting drug efficacy, toxicity, adverse reactions and selectivity (Carpentier and Dupont, 2000; Mirtallo et al., 2010; Soedirman et al., 1996).
The present study showed that the VK1 distribution in the body differs between injections of VK1–FE and VK1. After injection, plasma VK1 concentrations gradually decreased and the VK1 concentrations were much higher in the VK1 injection group than in the VK1–FE group, indicating that the VK1 distribution was faster in the VK1–FE group than in the VK1 injection group. There were significant differences in VK1 distribution between the two preparations. From a concentration perspective, the VK1 concentrations in the VK1–FE group in the heart and spleen were the highest; the second highest was in the liver, and the lowest were in the other organs. In the VK1 injection group, the liver VK1 concentration was the highest amongst all organs and tissues. The amounts of liver VK1 in both of the two preparations were the highest among all organs and tissues. Because muscle and fat have large masses, the amounts of VK1 in those organs were also high. The VK1 amounts in heart, spleen, lung, kidney and testis were low. However, the concentrations and amounts of VK1 in heart, spleen, lung and liver (at 4 h) in the VK1–FE group were much higher than in the VK1 injection group. To intuitively display the distribution of VK1 in the different organs and tissues, VK1 concentration–time curves were plotted (Fig. 11A). At 0 min, all of the VK1 was in the plasma, and the plasma VK1 concentration was calculated. Figure 11A shows that,
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Figure 11. The concentration–time (A) and amount–time (B) profiles of VK1–FE in the organs after 1 mg/kg VK1–FE intravenous injection. The data are given as the mean ± SEM, n = 4–9.
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Biomed. Chromatogr. 2015; 29: 1849–1858
Vitamin K1 distribution in rats after intravenous VK1–FE injection, the plasma VK1 was first transferred to the heart and spleen, then to the liver, and finally to the lung, kidneys, testis, muscles and fat. The amount of VK1 in the various organs and tissues were calculated based on their concentration and mass. The plasma, muscle and fat accounted for 4.1% (Zhou et al., 2012), 40% (Shao et al., 2012) and 4% (Duarte et al., 2012; Wen and Lee, 2014) of the body weight, respectively. The VK1 amounts in the different organs and tissues vs time curves were plotted (Fig. 11B). Figure 11(B) shows that the liver and muscle are the most important organ and tissue for VK1 distribution. VK1 is mostly stored in the liver (Marinova et al., 2013; Ronden et al., 1998), so the liver is the primary organ that affects VK1 distribution. In addition, this organ is important for eliminating VK1. The VK1 elimination half-life in patients with severe acute liver dysfunction is approximately twice that in normal individuals (Pereira et al., 2005). At 4 h, the VK1 concentration in the VK1–FE group was 1.6 times higher than that in the VK1 injection group, indicating that VK1–FE was more likely to concentrate in the liver. Our results suggest that VK1–FE is the better preparation. VK1 in the blood was initially transferred to the heart in the VK1–FE group. At 30 min, the VK1 concentration in the heart was the highest and was much higher than in the VK1 injection group. The concentration then rapidly declined. This suggested that heart has a high and temporary storage capacity for VK1–FE (Huber et al., 1999). However, it is unclear whether the heart has VK-dependent carboxylase. Fraser and Price (1988) found an amount of mRNA coding for VK-dependent matrix Gla protein in the heart. However, its function remains unclear. VK1 in the blood was secondarily transferred to the spleen in the VK1–FE group. The VK1 concentration in the spleen was lower than that in heart at 30 min and decreased more slowly than that in the heart. The VK1 concentrations in the heart and spleen were significantly higher in the VK1–FE group than in VK1 injection group. It is important to note that the heart and spleen were important organs that influenced the distribution differences between the two preparations. The VK1 concentrations and amounts in the lung, kidney and testis at all three time points were low and had little effect on the VK1 distribution in the rats. VK1 was not detected in the brain because it has difficulty crossing the blood–brain barrier. Rats receiving certain amounts of VK1 by dietary intake over several months have been found to have traces of it in the brain (Sato et al., 2003). In addition, VK1 distributed slowly in the muscle, fat and testis owing to the low blood flow in these tissues and organs. There were no significant differences in the VK1 concentrations in the muscle or fat between the two preparations, suggesting that these tissues did not contribute to the differences in VK1 distribution. Muscle accounts for approximately 40% of body weight (Shao et al., 2012). Although the VK1 concentration was low in muscle tissue, it may be a considerable VK1 storage tissue. Fat accounted for only 4% of the body weight in the rats. Because of its high lipid solubility, fat can accumulate VK1. Therefore, VK1 concentrations in fat gradually increased from 0.5 to 4 h. Based on the amounts detected, fat is an important tissue that affects the distribution of this vitamin.
Conclusion
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References Carpentier YA and Dupont IE. Advances in intravenous lipid emulsions. World Journal of Surgery 2000; 24: 1493–1497. Chakravarthy VA, Sailaja BB and Kumar AP. Stability-indicating RP-HPLC method for simultaneous estimation of enrofloxacin and its degradation products in tablet dosage forms. Journal of Analytical Methods in Chemistry 2015; 2015: 735145. Choonara IA, Scott AK, Haynes BP, Cholerton S, Breckenridge AM and Park BK. Vitamin K1 metabolism in relation to pharmacodynamic response in anticoagulated patients. British Journal of Clinical Pharmacology 1985; 20: 643–648. de Boer VC, Dihal AA, van der Woude H, Arts IC, Wolffram S, Alink GM, Rietjens IM, Keijer J and Hollman PC. Tissue distribution of quercetin in rats and pigs. Journal of Nutrition 2005; 135: 1718–1725. Duarte FO, Sene-Fiorese M, Cheik NC, Maria AS, de Aquino AE Jr, Oishi JC and Rossi EA. Garcia de Oliveira Duarte AC and Damaso AR. Food restriction and refeeding induces changes in lipid pathways and fat deposition in the adipose and hepatic tissues in rats with diet-induced obesity. Experimental Physiology 2012; 97: 882–894. Ducros V, Pollicand M, Laporte F and Favier A. Quantitative determination of plasma vitamin K1 by high-performance liquid chromatography coupled to isotope dilution tandem mass spectrometry. Analytical Biochemistry 2010; 401: 7–14. Fiore LD, Scola MA, Cantillon CE and Brophy MT. Anaphylactoid reactions to vitamin K. Journal of Thrombosis and Thrombolysis 2001; 11: 175–183. Fraser JD and Price PA. Lung, heart, and kidney express high levels of mRNA for the vitamin K-dependent matrix Gla protein. Implications for the possible functions of matrix Gla protein and for the tissue distribution of the gamma-carboxylase. Journal of Biological Chemistry 1988; 263: 11033–11036. Haroon Y and Hauschka PV. Application of high-performance liquid chromatography to assay phylloquinone (vitamin K1) in rat liver. Journal of Lipid Research 1983; 24: 481–484. Holmes MV, Hunt BJ and Shearer MJ. The role of dietary vitamin K in the management of oral vitamin K antagonists. Blood Review 2012; 26: 1–14. Huber AM, Davidson KW, O’Brien-Morse ME and Sadowski JA. Tissue phylloquinone and menaquinones in rats are affected by age and gender. Journal of Nutrition 1999; 129: 1039–1044. Marinova M, Lutjohann D, Breuer O, Kolsch H, Westhofen P, Watzka M, Mengel M, Stoffel-Wagner B, Hartmann G, Coch C and Oldenburg J. VKORC1-dependent pharmacokinetics of intravenous and oral phylloquinone (vitamin K1) mixed micelles formulation. European Journal of Clinical Pharmacology 2013; 69: 467–475. Mi YN, Ping NN, Xiao X, Zhu YB, Liu J and Cao YX. The severe adverse reaction to vitamin K1 injection is anaphylactoid reaction but not anaphylaxis. PLoS One 2014; 9: e90199. Mirtallo JM, Dasta JF, Kleinschmidt KC and Varon J. State of the art review: Intravenous fat emulsions: current applications, safety profile, and clinical implications. Annals of Pharmacotherapy 2010; 44: 688–700. Pereira SP, Rowbotham D, Fitt S, Shearer MJ, Wendon J and Williams R. Pharmacokinetics and efficacy of oral versus intravenous mixedmicellar phylloquinone (vitamin K1) in severe acute liver disease. Journal of Hepatology 2005; 42: 365–370. Plank RM, Steinmetz T, Sokal DC, Shearer MJ and Data S. Vitamin K deficiency bleeding and early infant male circumcision in Africa. Obstetrics and Gynecology 2013; 122: 503–505. Qiu S, Liu Z, Hou L, Li Y, Wang J, Wang H, Du W, Wang W, Qin Y and Liu Z. Complement activation associated with polysorbate 80 in beagle dogs. International Immunopharmacology 2013; 15: 144–149. Riegert-Johnson DL, Kumar S and Volcheck GW. A patient with anaphylactoid hypersensitivity to intravenous cyclosporine and subcutaneous phytonadione (vitamin K(1)). Bone Marrow Transplantation 2001; 28: 1176–1177.
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The liver is the primary organ for VK1 distribution. VK1–FE was found to be distributed more rapidly than VK1 in the rats. The VK1 amounts in the liver, heart, spleen and lung in the VK1–FE
group were higher than those in the VK1 injection group, while the plasma VK1 amounts in the VK1–FE group were lower than those in the VK1 group. In the VK1–FE group, VK1 was first transferred to the heart and spleen, then to the liver, and finally to other organs and tissues. In the VK1 injection group, it was first transferred to the liver and then to other organs and tissues.
X. Xiao et al. Ronden JE, Thijssen HH and Vermeer C. Tissue distribution of K-vitamers under different nutritional regimens in the rat. Biochimica Biophysica Acta 1998; 1379: 16–22. Sato T, Ozaki R, Kamo S, Hara Y, Konishi S, Isobe Y, Saitoh S and Harada H. The biological activity and tissue distribution of 2′,3′-dihydrophylloquinone in rats. Biochimica Biophysica Acta 2003; 1622: 145–150. Shao YJ, Zhu DC and Wu YM. Anatomicophysiology. People’s Medical Publishing House, 2012; 49. Soedirman JR, De Bruijn EA, Maes RA, Hanck A and Gruter J. Pharmacokinetics and tolerance of intravenous and intramuscular phylloquinone
(vitamin K1) mixed micelles formulation. British Journal of Clinical Pharmacology 1996; 41: 517–523. Wen CK and Lee TY. Electroacupuncture decreases the leukocyte infiltration to white adipose tissue and attenuates inflammatory response in high fat diet-induced obesity rats. Evidence Based Complementary and Alternative Medicine 2014; 2014: 473978. Zhou ZY, Xue ZM and Shao YX. Basic Course of Experimental Animal and Comparative Medicine. Soochow University Press, 2012; 170.
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Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015; 29: 1849–1858
Revised: 30 March 2015,
Accepted: 2 May 2015
Published online in Wiley Online Library: 2 June 2015
(wileyonlinelibrary.com) DOI 10.1002/bmc.3506
Vitamin K1 distribution following intravenous vitamin K1–fat emulsion administration in rats Xue Xiaoa, Yan-Ni Mia, Fa Wangb, Bing-Hua Zhangb, Lei Caoa* and Yong-Xiao Caoa ABSTRACT: This study investigated vitamin K1 (VK1) distribution following intravenous vitamin K1–fat emulsion (VK1–FE) administration and compared it with that after VK1 injection. Rats were intravenously injected with VK1–FE or VK1. The organ and tissue VK1 concentrations were determined using high-performance liquid chromatography method at 0.5, 2 and 4 h to determine distribution, equilibrium and elimination phases, respectively. In the VK1–FE group, the plasma, heart and spleen VK1 concentrations decreased over time. However, other organs like liver, lung, kidney, muscle and testis, reached peak VK1 concentrations at 2 h. In the VK1 injection group, the liver VK1 concentrations were significantly higher than those in other organs at the three time points. However, VK1 concentrations in the other organs peaked at 2 h. In addition, in VK1–FE group, the heart, spleen and lung VK1 concentrations were significantly higher than those in the VK1 injection group at the three time points, and the liver VK1 concentration was significantly higher than that in the VK1 injection group at 4 h. The VK1 amount was greatest in the liver compared with the other organs. Thus, the liver is the primary organ for VK1 distribution. The distribution of VK1 is more rapid when injected as VK1–FE than as VK1. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: vitamin K1; vitamin K1-fat emulsion; distribution; high-performance liquid chromatography
Introduction Vitamin K1 (VK1) is essential for the formation of prothrombin and coagulation factors II, VII, IX and X. These factors are glycoproteins with a number of γ-carboxyglutamic acid residues clustered at the N-terminal end of the peptide chain (Holmes et al., 2012). VK1 also acts on proteins C and S, as well as non-coagulation proteins, such as osteocalcin and matrix Gla protein, which are involved in the metabolic pathways of bone and other tissues (Marinova et al., 2013). VK1 is mainly used to treat and/or prevent VK1 deficiency, specifically in newborns (Plank et al., 2013), and to antagonize the effects of warfarin (Holmes et al., 2012). Recently, the number of severe adverse reactions caused by the intravenous administration of VK1 has increased and attracted public attention (Fiore et al., 2001; Riegert-Johnson et al., 2001). Because VK1 is a lipid-soluble substance and is poorly soluble in water, a solubilizer, such as Tween 80, is required to prepare it for injection (Qiu et al., 2013). However, Tween 80 as a drug accessory has recently been implicated in severe adverse clinical effects. To avoid using a solubilizer, a new vitamin K1–fat emulsion (VK1–FE) preparation has been developed (Mi et al., 2014). We speculated that VK1–FE could change the distribution characteristics of VK1 and that the VK1 concentrations in some tissues of intravenously VK1–FE-injected animals could be changed. The objective of the present study was to investigate the VK1 distribution after intravenous VK1–FE administration and to compare it with that after VK1 injection to illustrate the VK1 distribution characteristics of these two preparations in rats.
Materials and methods
(Xi’an, China), respectively. VK1 standard was purchased from the National Institute for Food and Drug Control (Beijing, China), and its purity was >98% (high-performance liquid chromatography, HPLC). The injectable heparin sodium was purchased from Xingmao Co. (Shanghai, China). Ethanol and cyclohexane were obtained from Kemiou Reagent Co. (Tianjin, China) and were of HPLC grade.
Animals and drug administration Sprague–Dawley rats weighing 200–220 g and Kunming mice weighing 18–22 g (half males and half females) were purchased from the Animal Center of Xi’an Jiaotong University (Xi’an, China). The animals were maintained in conventional laboratory conditions for temperature, humidity and light and allowed food (standard pellet diet) and tap water ad libitum. The study was approved by the Ethics Committee at Xi’an Jiaotong University College of Medicine. Sixty mice were randomly divided into 10 groups, and a single dose of 2 mg/kg VK1–FE was injected into mice via the tail vein. The livers were collected at 0, 0.17, 0.5, 1, 2, 4, 6, 8, 10 and 12 h after the injection (Choonara et al., 1985). The rats were fasted for 24 h, but had free access to water. Then, the rats were randomly divided * Correspondence to: Lei Cao, Department of Pharmacology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi, 710061, China. Email: [email protected] a
Department of Pharmacology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi, 710061, China
b
Shaanxi Institute for Food and Drug Control, Xi’an, Shaanxi, 710061, China
Drugs and reagents
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Abbreviations used: VK1, vitamin K1; VK1–FE, vitamin K1–fat emulsion.
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The injectable VK1–FE and VK1 were purchased from Cisen Pharmaceutical Co. (Shandong, China) and Anjian Pharmaceutical Co.
X. Xiao et al. into three groups: the control group (n = 8); the VK1–FE group (n = 27); and the VK1 injection group (n = 27). Rats from the latter two groups were injected with a single dose (1 mg/kg) of VK1–FE or VK1 via the small saphenous vein. The blood, organ and tissue samples, such as the heart, liver, spleen, lung, kidneys, brain, testis, muscle and fat, were collected at 0.5 and 4 h after injection (de Boer et al., 2005). Instruments and chromatographic conditions Chromatographic analyses were performed under ambient conditions with a Waters Alliance HT HPLC system (Waters Association, Milford, MA). This system is a ZMPOWER-2 chromatography workstation. It has a variable wavelength programmable UV spectrophotometric detector (Waters 2489), which was operated at 254 nm, and an automated injection system. Chromatographic separations were achieved using a Kromasil 100-5 C18 column (150 × 4.6 mm, o.d., 8 mm, Akzonobel, Sweden). The mobile phase consisted of ethanol–water–diethyl ether (88:2:10, v/v) at a flow rate of 1.5 mL/min. The column oven temperature was 30 °C, and the injection volume was 50 μL. Blood and tissue sample preparation Blood samples were collected from the abdominal aorta, transferred into a 5 mL lithium heparin anticoagulant tube and immediately protected from light. Then, plasma was obtained by centrifugation (2500 rpm/10 min). One milliliter of plasma was deproteinized with 3 mL absolute ethyl alcohol, and then 3 mL cyclohexane was added and mixed vigorously. The mixture was centrifuged (12,000 rpm/5 min), and then the cyclohexane was removed and evaporated. To obtain the residue, 1 mL absolute ethyl alcohol was added, mixed vigorously and centrifuged twice for 5 min at 12,000 rpm, and 50 μL of the supernatant was injected into the HPLC column. The whole heart, pancreas, kidneys, lungs, testis, brain and 2.0 g liver, 2.0 g thigh muscle and 1.0 g fat from around the kidney were removed from the rats. The collected organs and tissues were cleaned, dried with filter paper and then weighed. Each organ or tissue was added to 3 mL 75% ethanol and homogenized using a Polytron homogenizer. Each homogenate was added to 3 mL cyclohexane and centrifuged. The solvent layers were isolated and dried. The resultant pellet was dissolved in 1 mL absolute ethyl alcohol, mixed vigorously, centrifuged twice for 5 min at 12,000 rpm, and then analyzed by HPLC. All of the experimental processes were performed in the dark (Qiu et al., 2013). Assay method validation To evaluate the performance of the developed extraction method, analytical parameters, including linearity, limit of detection (LOD), limit of quantification (LOQ), reproducibility, stability, recovery and precision were investigated. Protein precipitation. To develop a suitable and efficient method for VK1 extraction from rat liver, we used absolute alcohol and physiological saline for protein precipitation. Liver samples containing significantly less VKl were divided into four groups (four samples per group). The ratios of absolute alcohol to physiological saline were 4:1, 3:1, 2:1 and 1:1 (v/v). A chromatogram was recorded (Haroon and Hauschka, 1983).
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Linearity and LOD and LOQ. Blank blood and tissue samples were prepared by homogenizing fresh blood, heart, liver, spleen,
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lung, kidney, testis, muscle or fat in 75% ethanol. Linearity was evaluated within the concentration ranges of 0–100 μg/mL VK1 spiked into blank plasma and tissues. The LOD is considered the concentration that has a signal-to-noise ratio (S/N) of 3:1. The LOQ is the lowest concentration that has an S/N ratio of at least 10:1. Reproducibility. Reproducibility is defined as the variation that occurs when different analysts measure the same samples with the same procedure in different facilities. We used liver as a representative sample. Three identical samples, containing a specific amount of VK1 (1 μg/mL), were analyzed in separate HPLC runs, using the same procedure in all three runs but operated by different analysts, and the peak area of VK1 was detected. Reproducibility was determined as percentage relative standard deviation (RSD). Stability. To ensure the reliability of the results in relation to the handling and storing of plasma and tissue samples, stability studies were performed. Liver samples containing 1.0 μg/mL VK1 were prepared, and the VK1 concentrations were measured at 0, 2, 4, 6, 8, 10 and 12 h. Recovery. The recovery of VK1 from plasma and other tissues was determined at concentrations of 0.4, 2 and 10 μg/mL. Three replicates of each concentration were injected into the HPLC system. The extraction recovery at each concentration was calculated using the following equation: Recovery ð%Þ ¼ ða=bÞ100% where a is the peak area obtained from the spiked samples and b is the quantity of the spiked VK1 added. Precision. The precision of this method was assessed by adding a quantity of VK1 (2 μg/mL) to liver samples and measuring VK1 concentration using the assay procedure. Seven standard samples were prepared and assayed separately. The relationship between VK1 concentration and time in mouse liver We speculated that the liver is a major target organ of VK1 distribution. To determine the time points of VK1 distribution, the body processing of VK1 was predicted by measuring the VK1 concentration vs time curves in mouse livers. The mice were randomly divided into 10 groups (six mice per group). A single dose of 2 mg/kg VK1–FE was injected into mice via the tail vein. After 0, 0.17, 0.5, 1, 2, 4, 6, 8, 10 and 12 h, the mice were sacrificed by CO2 asphyxiation. In a dark room, the livers were removed and cleared with 0.9% (w/v) sodium chloride. The livers were dried, weighed, transferred into tubes with 1 mL 0.9% sodium chloride and 3 mL absolute alcohol, and then homogenized for 3 min using a blender. Three milliliters of cyclohexane were added and mixed vigorously. Then, the mixture was centrifuged. The cyclohexane layer was removed and evaporated (Sato et al., 2003). The residues were stored in the dark until analyzed. Tissue distribution of VK1 After 24 h of fasting, a single dose of 1 mg/kg VK1–FE or VK1 was intravenously injected into the rats. Before (n = 8) and 0.5, 2 and 4 h (n = 9) after drug injection, surgeries were performed under chloral hydrate anesthesia. A blood sample was collected from the abdominal aorta and transferred into a 5 mL lithium heparin
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Vitamin K1 distribution in rats anticoagulant tube. Plasma was obtained after centrifugation (2500 rpm/10 min) (Ducros et al., 2010). Then, the heart, liver, spleen, lungs, kidneys, brain, testis, muscle and fat were removed, cleared, dried and weighed. The tissue sample preparation procedure was the same as detailed in ’Blood and tissue sample preparation’.
Therefore, absolute alcohol–physiological saline at a 3:1 ratio was chosen as the extraction solvent for the VK1 assay. Linearity, LOD and LOQ. Under the chromatographic conditions used in this experiment, the retention time of VK1 was approximately 2.93 min (Fig. 1). The VK1 regression models in organs or tissues were represented by the following equation: y ¼ ax þ b
Data analysis Linear regression calculations were performed using Microsoft Excel 2010. The statistical analyses were performed using GRAPHPAD PRISM 5.0 (GraphPad Software, La Jolla, CA, USA). The results are presented as the mean ± SEM, and p < 0.05 was considered statistically significant. Student’s t-test was used to compare the VK1 concentration between the VK1–FE and VK1 injection groups.
The x-axis represents the VK1 concentration, and the y-axis represents the VK1 peak area. The correlation coefficient (r 2) indicates sufficient linearity. The r2 values in the nine organs and tissues ranged from 0.9980 to 1.0000. The LOQ, 10 times the S/N ratio, was 32 ng/mL. The LOD, 3 times the S/N ratio, was 14 ng/mL (Chakravarthy et al., 2015). Reproducibility. Three VK1 standard samples were analyzed in separate HPLC runs using the same procedure in all three runs and operated by three different analysts, which resulted in an RSD for the VK1 peak area of 1.682%. This result indicated that this method was consistent with the experimental requirements.
Results Assay method validation Protein precipitation. In pilot experiments, absolute alcohol, acetonitrile and methanol were selected for protein precipitation. We found that absolute alcohol produced the best results. Our purpose was to determine the optimal ratio of absolute alcohol and physiological saline for protein precipitation. The results demonstrated that the highest VK1 concentration was 4.12 ± 0.53 μg/g when the ratio was 3:1, which was considered to be the most suitable and efficient method for VK1 extraction.
Stability. Liver samples containing 1.0 μg/mL VK1 were prepared and assayed. The variation was calculated by the following equation: Variation ð%Þ ¼ ½ðx y Þ = y 100% where x is the peak area at the time point measured and y is the peak area at 0 h. Table 1 shows that the variation ranged from 8.5 to 2.4%.
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Figure 1. HPLC chromatograms of vitamin K1 (VK1). Representative chromatograms of tissues or organs at 2 h after intravenous vitamin K1–fat emulsion (VK1–FE) administration. Each chromatogram includes VK1 standard solution (A), plasma (B), heart (C), liver (D), spleen (E), lung (F), kidney (G), muscle (H), fat (I) and testis ( J).
X. Xiao et al. Table 1. vitamin K1 (VK1) stability in rat livers Detected time (h)
Peak area (μV s)
Variation (%)
41,518 41,827 41,339 42,325 42,516 41,200 41,165
— 0.74 0.43 1.94 2.4 –0.76 –0.85
0 2 4 6 8 10 12
Recovery and precision. The recovery of VK1 from different organs or tissues was determined at concentrations of 0.4, 2 and 10 μg/mL. The results are shown in Table 2. The coefficient of variation for the three concentration samples ranged from 62.76 to 102.11%. The RSD ranged from 0.03 to 2.65%. The precision of this method was assessed by adding VK1 (2 μg/mL) to seven liver samples. VK1 concentrations were determined by the assay procedure, and the RSD was 0.57%. VK1 distribution in mouse liver VK1 concentration–time curve in mouse livers. We hypothesized that the liver is the major target organ of VK1 distribution. To determine the time points of VK1 distribution, VK1 present in the body was predicted by measuring its concentrations in mouse livers. Curves of VK1 concentration vs time were then constructed. The results are shown in Fig. 2. The VK1 concentration peaked (maximum of 11.00 ± 1.44 μg/g) at 2 h after VK1–FE intravenous administration, and then it consistently declined. At 12 h, the concentration was close to the level at 0 h. From the VK1 concentration–time curve, we selected 0.5, 2 and 4 h to represent the distribution, equilibrium and elimination phases of VK1, respectively. VK1 distribution in rats There were no significant differences in body weight or organ weights in the rats between the VK1 injection group and the VK1–FE group at the three time points (Table 3). After 24 h of fasting, VK1 was not detected in any of the organs or tissues. Distribution phase. Thirty minutes after administration was selected to represent the distribution phase of VK1. VK1 distribution Table 2. VK1 recovery in the plasma and other tissues or organs (n = 3–5, mean ± SEM) The recovery of different VK1 concentrations (%)
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Plasma Heart Liver Spleen Lung Kidney Muscle Fat Testis
0.4 μg/mL
2 μg/mL
10 μg/mL
99.34 ± 0.54 82.72 ± 0.44 88.61 ± 1.12 67.27 ± 1.10 92.09 ± 0.55 89.63 ± 0.34 84.81 ± 0.28 67.16 ± 1.53 89.70 ± 1.56
102.11 ± 0.60 81.32 ± 0.74 84.24 ± 0.16 63.27 ± 0.02 91.89 ± 0.02 85.74 ± 0.08 86.85 ± 0.85 64.99 ± 0.05 92.03 ± 0.07
100.58 ± 0.46 83.00 ± 0.02 88.13 ± 0.19 70.15 ± 0.41 91.88 ± 0.10 86.16 ± 0.98 88.40 ± 0.47 62.76 ± 0.07 93.81 ± 1.60
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Figure 2. Liver VK1 amount–time profile following the injection of a single dose of VK1–FE in mice. The data are given as the mean ± SEM, n = 8.
in various organs and tissues were determined. First, VK1 concentrations (μg/g) were measured in different organs and tissues. Then, the amounts of VK1 (μg) in the various organs were calculated according to their weights. The results showed that, in the VK1 injection group, the concentration and amount of VK1 in the liver were higher than in the other organs, including the heart, spleen, lung, kidney, muscle, fat, testis and blood. In the VK1–FE group, the concentration and/or amount of VK1 in the heart, liver, and spleen were higher than those in the other organs and tissues, such as the lung, kidney, muscle, fat and testis. The concentrations and amounts of VK1 in the heart, spleen, and lung in the VK1–FE group were significantly higher than those of the VK1 injection group (p < 0.01). However, the concentrations and the total amount of VK1 in the liver, kidney, muscle, fat, and testis were not significantly different between the two preparations (p > 0.05; Fig. 3). Equilibrium phase. Two hours after drug administration was chosen to represent the equilibrium phase. In the VK1 injection group, the concentration and amount of VK1 in liver were higher than those in other organs and tissues, such as the blood, heart, spleen, lung, kidney, muscle, fat and testis. In the VK1–FE group, the concentrations and/or amounts of VK1 were higher in the heart, liver and spleen than those in the other organs and tissues, such as the lung, kidney, muscle, fat, testis and plasma. However, it was not detected in the brain in either the VK1–FE or the VK1 injection group. The VK1 concentrations and amounts in the heart, spleen and lung in the VK1–FE group were significantly higher than those in the VK1 injection group (p < 0.01), while the VK1 concentration in plasma was significantly lower than in the VK1 injection group (p < 0.05). There was no significant difference in the VK1 concentrations and/or the amounts in the liver, spleen, muscle and testis between the two preparations (p > 0.05; Fig. 4). Elimination phase. Four hours after drug administration was selected to represent the elimination phase. The results showed that the VK1 concentrations in the plasma, heart, liver, spleen, lung, kidney and muscle at 4 h were lower than those at 2 h in both the VK1 injection group and VK1–FE group. The
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Vitamin K1 distribution in rats Table 3. Rat body and organ weights in the VK1 and VK1–FE injection groups (n = 4–9, mean ± SEM) Weight at 30 min ( g)
Weight Heart Liver Spleen Lung Kidney Testis
Weight at 2 h ( g)
Weight at 4 h ( g)
VK1–FE
VK1
VK1–FE
VK1
VK1–FE
VK1
200 ± 2.7 0.66 ± 0.02 7.20 ± 0.17 0.52 ± 0.04 1.11 ± 0.02 2.06 ± 0.01 2.27 ± 0.11
198 ± 4.3 0.67 ± 0.02 7.10 ± 0.17 0.53 ± 0.02 1.03 ± 0.02 1.89 ± 0.05 2.25 ± 0.05
199 ± 2.7 0.67 ± 0.02 7.88 ± 0.15 0.50 ± 0.03 1.13 ± 0.03 1.93 ± 0.08 2.11 ± 0.16
198 ± 1.7 0.67 ± 0.03 8.28 ± 0.22 0.50 ± 0.03 1.12 ± 0.03 1.88 ± 0.04 2.27 ± 0.16
206 ± 2 0.71 ± 0.01 9.12 ± 0.23 0.61 ± 0.22 1.17 ± 0.06 1.83 ± 0.03 1.93 ± 0.05
204 ± 2 0.68 ± 0.02 8.81 ± 0.51 0.57 ± 0.03 1.10 ± 0.03 1.73 ± 0.04 1.91 ± 0.11
Figure 3. The concentrations (A) and amounts (B) of VK1 in the organs at 30 min after 1 mg/kg intravenous administration of VK1–FE or VK1 in rats. The data are given as the means ± SEM. Heart and lung, n = 9; liver and spleen, n = 8; kidney, n = 6; testis, n = 4. **p < 0.01 vs the VK1 injection group.
Figure 4. The concentrations (A) and amounts (B) of VK1 in the organs at 2 h after 1 mg/kg intravenous administration of VK1–FE or VK1. The data are given as the mean ± SEM. Blood and lung, n = 9; heart, liver and spleen, n = 8; kidney, n = 6; testis, n = 4. **p < 0.01 vs the VK1 injection group.
liver VK1 concentration decreased rapidly in the VK1 injection group. The concentrations and amounts of VK1 in the heart, spleen, lung and liver remained significantly higher in the VK1–FE group than in the VK1 injection group (p < 0.01). There was no significant difference in the VK1 concentrations and amounts in the kidney, muscle, fat and testis between the VK1–FE and VK1 injection groups (p > 0.05; Fig. 5). Distribution changes of VK1 in various organs and tissues over time
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Liver. The liver VK1 concentration was not the highest among the organs in the VK1–FE group (Figs. 3A, 4A and 5A). Because
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Plasma. Before the intravenous administration of VK1, it was not detected in the plasma. In the VK1 injection group, the plasma VK1
concentrations at 0.5, 2 and 4 h were 0.92 ± 0.20, 0.26 ± 0.03 and 0.04 ± 0.01 μg/mL, respectively, indicating a gradual decrease in the concentration. Thirty minutes after VK1–FE administration, the VK1 concentration was 0.45 ± 0.15 μg/mL, which was approximately half of the concentration in the VK1 injection group at this time point. The VK1 concentrations were 0.11 ± 0.04 and 0.06 ± 0.01 μg/mL at 2 and 4 h, respectively, in the VK1–FE group. At 2 h, the plasma VK1 concentration in the VK1 injection group was 2.3-fold higher than that in the VK1–FE group. Although the plasma VK1 concentration decreased over time, the reduction was more rapid in the VK1 injection group than in the VK1–FE group.
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Figure 5. The concentrations (A) and amounts (B) of VK1 in the organs at 4 h after 1 mg/kg intravenous administration of VK1–FE or VK1. The data are given as the mean ± SEM. Blood, heart, spleen and lung, n = 9; liver, n = 8; kidney, n = 6; testis, n = 4. **p < 0.01 vs the VK1 injection group.
the mass of the liver is large, the amount of VK1 in the liver was the highest amongst the organs (Figs. 3B, 4B and 5B), suggesting that the liver is the most important organ affecting VK1 distribution. Figure 6 shows the changes in VK1 concentrations and amounts in the liver over time. Thirty minutes after the intravenous injection of VK1–FE or VK1, the VK1 concentrations in the livers were at the same level, 2.27 ± 0.39 or 2.32 ± 0.28 μg/g, respectively (p > 0.05). At 2 h, the VK1 concentrations in the VK1–FE and VK1 injection groups were 3.60 ± 0.69 and 3.34 ± 0.46 μg/g, respectively (p > 0.05), which were higher than those at 30 min. The VK1 concentration was significantly higher in the VK1–FE group than in the VK1 injection group at 4 h (p 0.05). Brain and testis. The testis VK1 concentrations and amounts at the three time points were very low in the two preparations and had no impact on its distribution. VK1 was not detected in the brain at the three time points.
Discussion An emulsion carrier can increase the water solubility of a poorly soluble compound, changing the distribution of the drug in the body, increasing the drug permeability of biological membranes and affecting drug efficacy, toxicity, adverse reactions and selectivity (Carpentier and Dupont, 2000; Mirtallo et al., 2010; Soedirman et al., 1996).
The present study showed that the VK1 distribution in the body differs between injections of VK1–FE and VK1. After injection, plasma VK1 concentrations gradually decreased and the VK1 concentrations were much higher in the VK1 injection group than in the VK1–FE group, indicating that the VK1 distribution was faster in the VK1–FE group than in the VK1 injection group. There were significant differences in VK1 distribution between the two preparations. From a concentration perspective, the VK1 concentrations in the VK1–FE group in the heart and spleen were the highest; the second highest was in the liver, and the lowest were in the other organs. In the VK1 injection group, the liver VK1 concentration was the highest amongst all organs and tissues. The amounts of liver VK1 in both of the two preparations were the highest among all organs and tissues. Because muscle and fat have large masses, the amounts of VK1 in those organs were also high. The VK1 amounts in heart, spleen, lung, kidney and testis were low. However, the concentrations and amounts of VK1 in heart, spleen, lung and liver (at 4 h) in the VK1–FE group were much higher than in the VK1 injection group. To intuitively display the distribution of VK1 in the different organs and tissues, VK1 concentration–time curves were plotted (Fig. 11A). At 0 min, all of the VK1 was in the plasma, and the plasma VK1 concentration was calculated. Figure 11A shows that,
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Figure 11. The concentration–time (A) and amount–time (B) profiles of VK1–FE in the organs after 1 mg/kg VK1–FE intravenous injection. The data are given as the mean ± SEM, n = 4–9.
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Vitamin K1 distribution in rats after intravenous VK1–FE injection, the plasma VK1 was first transferred to the heart and spleen, then to the liver, and finally to the lung, kidneys, testis, muscles and fat. The amount of VK1 in the various organs and tissues were calculated based on their concentration and mass. The plasma, muscle and fat accounted for 4.1% (Zhou et al., 2012), 40% (Shao et al., 2012) and 4% (Duarte et al., 2012; Wen and Lee, 2014) of the body weight, respectively. The VK1 amounts in the different organs and tissues vs time curves were plotted (Fig. 11B). Figure 11(B) shows that the liver and muscle are the most important organ and tissue for VK1 distribution. VK1 is mostly stored in the liver (Marinova et al., 2013; Ronden et al., 1998), so the liver is the primary organ that affects VK1 distribution. In addition, this organ is important for eliminating VK1. The VK1 elimination half-life in patients with severe acute liver dysfunction is approximately twice that in normal individuals (Pereira et al., 2005). At 4 h, the VK1 concentration in the VK1–FE group was 1.6 times higher than that in the VK1 injection group, indicating that VK1–FE was more likely to concentrate in the liver. Our results suggest that VK1–FE is the better preparation. VK1 in the blood was initially transferred to the heart in the VK1–FE group. At 30 min, the VK1 concentration in the heart was the highest and was much higher than in the VK1 injection group. The concentration then rapidly declined. This suggested that heart has a high and temporary storage capacity for VK1–FE (Huber et al., 1999). However, it is unclear whether the heart has VK-dependent carboxylase. Fraser and Price (1988) found an amount of mRNA coding for VK-dependent matrix Gla protein in the heart. However, its function remains unclear. VK1 in the blood was secondarily transferred to the spleen in the VK1–FE group. The VK1 concentration in the spleen was lower than that in heart at 30 min and decreased more slowly than that in the heart. The VK1 concentrations in the heart and spleen were significantly higher in the VK1–FE group than in VK1 injection group. It is important to note that the heart and spleen were important organs that influenced the distribution differences between the two preparations. The VK1 concentrations and amounts in the lung, kidney and testis at all three time points were low and had little effect on the VK1 distribution in the rats. VK1 was not detected in the brain because it has difficulty crossing the blood–brain barrier. Rats receiving certain amounts of VK1 by dietary intake over several months have been found to have traces of it in the brain (Sato et al., 2003). In addition, VK1 distributed slowly in the muscle, fat and testis owing to the low blood flow in these tissues and organs. There were no significant differences in the VK1 concentrations in the muscle or fat between the two preparations, suggesting that these tissues did not contribute to the differences in VK1 distribution. Muscle accounts for approximately 40% of body weight (Shao et al., 2012). Although the VK1 concentration was low in muscle tissue, it may be a considerable VK1 storage tissue. Fat accounted for only 4% of the body weight in the rats. Because of its high lipid solubility, fat can accumulate VK1. Therefore, VK1 concentrations in fat gradually increased from 0.5 to 4 h. Based on the amounts detected, fat is an important tissue that affects the distribution of this vitamin.
Conclusion
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The liver is the primary organ for VK1 distribution. VK1–FE was found to be distributed more rapidly than VK1 in the rats. The VK1 amounts in the liver, heart, spleen and lung in the VK1–FE
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