Menaquinone-4 Concentration Is Correlated with Sphingolipid Concentrations in Rat Brain1

  1. Guylaine Ferland2
  1. Département de Nutrition, Université de Montréal, H3C 3J7 Montréal, Canada;
  2. *INSERM U346, Dermatologie, Hôpital Edouard Herriot, Lyon, France and
  3. †Douglas Hospital Research Center, Department of Psychiatry, McGill University, Montréal, Canada
  1. ↵2To whom correspondence should be addressed. E-mail: Guylaine.Ferland{at}umontreal.ca.
 
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Abstract

Studies with animals support a role for vitamin K (VK) in the biosynthesis of sphingolipids, a class of complex lipids present in high concentrations in the brain. In mice and rats, VK deficiency decreases levels of brain sulfatides and causes behavioral alterations. In light of its heterogeneity and to better understand the role of VK in the brain, we characterized the distribution of the two main VK vitamers, phylloquinone (K1) and menaquinone-4 (MK-4), in nine distinct brain regions. Weaning female Sprague-Dawley rats (n = 5/dietary group) were fed diets containing either low (L, 80 μg/kg diet), adequate (A, 500 μg/kg diet) or high (H, 2000 μg/kg diet) levels of K1 for 6 mo. The main form of VK in the brain was MK-4, and it was present in significantly higher concentrations in myelinated regions (the pons medulla and midbrain) than in nonmyelinated regions. Both regional K1 and MK-4 increased with K1 intake (P < 0.05). Sphingolipid distribution varied across brain regions (P < 0.001) but was not affected by K1 intake. In the L and A groups but not the H group, brain MK-4 concentration was positively correlated with the concentrations of sulfatides (L, r = 0.518; A, r = 0.479) and sphingomyelin (L, r = 0.515; A, r = 0.426), and negatively correlated with ganglioside concentration (L, r = −0.398); A, r = −0.353). Sphingolipids are involved in major cellular events such as cell proliferation, differentiation and survival. The strong associations reported here between brain MK-4 and sphingomyelin, sulfatides and gangliosides suggest that this vitamer may play an important role in the brain.

Vitamin K (VK)2 is classically known for its role in the biological activation of a family of proteins involved in hemostasis, bone metabolism, tissue calcification and cell cycle regulation (1,2). However, a lesser known function of the vitamin is its participation in the synthesis of sphingolipids, a class of complex lipids found in high concentrations in the brain that includes cerebrosides, sphingomyelin, sulfatides, ceramides and gangliosides (3,4). Cerebrosides, sphingomyelin and sulfatides are usually associated with the white matter and the myelin sheath, whereas ceramides and gangliosides are markers of neuronal membranes and are associated with the gray matter (5,6). Although originally appreciated for their role as essential structural components of cell membranes, sphingolipids are now known to participate in important cellular events such as cell signaling (7), proliferation, differentiation and survival (8–10).

A role for VK in sphingolipid metabolism was first established in bacteria (11) and then in mice when it was observed that feeding warfarin, a VK antagonist, to young mice decreases brain concentrations of sulfatide (12) and galactocerebroside sulfotransferase (GST), the main sulfatide synthetic enzyme (13). In both cases, feeding phylloquinone (K1) reversed the effect of warfarin. In later studies, feeding mice an excess of K1 markedly increased brain sulfatide concentration and GST activity (14). More recently, mice with a deficiency in sulfatides and cerebrosides generated by knocking out the ceramide galactosyltransferase enzyme gene presented several neurological abnormalities, including dysmyelinosis, loss of axonal conduction velocity, constant body tremor, seizures and loss of locomotor activity, and died by 3 mo of age (15,16). In an independent report, VK deficiency induced by dietary depletion or by warfarin treatment was associated with hypoactivity and a lack of exploratory behavior in rats (17).

Reports published in the past decade confirm the presence of VK in brain homogenates in concentrations that generally reflect intake (18–21). Interestingly, although VK is present in the forms of K1 and menaquinone-4 (MK-4) in the majority of extrahepatic tissues, in the brain VK is present predominantly as MK-4. Explanations for this tissue-specific vitamer profile are not yet available but could suggest a unique role for MK-4 in the brain (22,23). The brain is a very heterogeneous entity with distinct anatomical characteristics and functions. To better understand the role of VK in the brain and in sphingolipid metabolism in particular, we characterized the distribution of the K1 and MK-4 vitamers in nine distinct brain regions of 6-mo-old female Sprague-Dawley rats fed different amounts of K1 since weaning, and related their regional tissue concentrations to those of sphingolipids.

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MATERIALS AND METHODS

Animals and diets.

This study was approved by the Animal Care Committee of the University of Montreal in compliance with the guidelines of the Canadian Council on Animal Care. Female Sprague-Dawley rats (age 4 wk, n = 15) were obtained from Charles Rivers Canada (St Constant, QC) and housed in groups of three in suspended stainless steel wire-bottom cages in a room kept at 22°C with a 12-h light–dark cycle. The rats had free access to water and food. The rats were fed a standard rat diet for a 1-wk acclimatization period and were then randomly assigned to an AIN-76–based diet (24) containing low (L; 80 μg/kg), adequate (A; 500 μg/kg) or high (H; 2000 μg/kg) concentrations of K1 (Sigma Chemicals, St Louis, MO). There were 5 rats per dietary group. The powdered diets were prepared in the laboratory and their K1 concentrations were assayed by HPLC analysis as follows (means ± sem, n = 8): L, 90.7 ± 7.5; A, 513.5 ± 15.0 and H, 2022.6 ± 59.9 μg/kg. Food intake and body weight were recorded every 2 wk and did not differ among groups.

At 6 mo of age, the rats were anesthetized with pentobarbital and killed by bleeding from the abdominal aorta. Whole brains were quickly removed and dissected on ice into cerebellum, midbrain, pons medulla, frontal cortex, olfactory bulb, hypothalamus, thalamus, hippocampus and striatum. After dissection, the tissue samples were frozen immediately in liquid nitrogen and stored at −70°C until analysis.

Vitamin K analysis.

The K1 and MK-4 were extracted and quantified as described by Huber et al. (21). In short, tissue samples were pulverized in anhydrous Na2SO4 (10 × tissue weight) and extracted for 1 h in 10 mL of acetone containing an internal standard [2′,3′-dihydrophylloquinone (K1H2) at 2.2 pmol in 50 μL; a gift from the Vitamin K laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA]. Extracts were centrifuged (500 × g for 10 min at 4°C) and dried under nitrogen at 45°C, then the solid residue was reextracted with 6 mL of hexane and 2 mL of water for 3 min. After centrifugation, the top hexane layer was dried under nitrogen and redissolved in 2 mL of hexane for solid phase extraction on 3-mL silica gel columns (JT Baker, Phillipsburg, NJ). The K1 and MK-4 fraction was eluted with 8 mL of hexane:diethyl ether (97:3, v/v) and evaporated under nitrogen, then the residue was dissolved in 0.02 mL of dimethyl chloride and 0.180 mL methanol containing an aqueous phase (10 mmol/L zinc chloride, 10 mmol/L acetic acid and 5 mmol/L sodium acetate; 5 mL of the aqueous phase was added to 1 L of methanol). Quantitative analysis of the vitamers was performed by reverse-phase HPLC using a C-18 reverse-phase column and fluorescence detection (Waters, Mississauga, ON). The calibration standard consisted of a mixture of K1, MK-4 and K1H2 at 2.2 pmol in 50 μL. The recovery percentage for the samples was 85 to 90%, calculated from the internal standard.

Sphingolipid analyses.

The different sphingolipid classes (gangliosides, ceramides, cerebrosides, sulfatides and sphingomyelin) were purified by solid-phase extraction according to the method of Bodennec et al. (25,26). All solvents were analytical grade and were purchased from Fischer Scientific Canada (Nepean, ON). Sphingoid bases used as standards (cerebroside sulfate, sialic acid and sphingosine) were from Sigma (St Louis, MO). Lipids were extracted from the brain regions using chloroform:methanol (2:1, v/v) and partitioned according to the method of Folch et al. (27). Gangliosides, contained in the upper phase, were eluted according to the method of Williams & McCluer (28). After evaporation, gangliosides were suspended in chloroform:methanol (2:1, v/v). Total gangliosides were measured by the quantification of free sialic acids according to the method of Jourdian et al. (29). The Folch lower phase, containing ceramides, cerebrosides, sulfatides and sphingomyelin, was evaporated and resuspended in 500 μL of chloroform and loaded onto 500-mg LC-NH2 columns (Supelco, Oakville, ON) preconditioned with 2 mL of hexane. The cartridges were washed with 2 mL of ethyl ether. Ceramides were eluted with 3 mL of chloroform:methanol (23:1, v/v). Columns were then washed with 2 mL of diisopropyl ether:acetic acid (98:5, v/v). Cerebrosides were eluted with 4 mL of acetone:methanol (9:1.35, v/v). Sphingomyelin was eluted with 3 mL of chloroform:methanol (2:1, v/v). Finally, sulfatides were obtained by washing with 4 mL of methanol containing 0.2 mol/L aqueous ammonium acetate. This fraction was then applied to a C-18 silica column preconditioned with 3 mL of PBS:methanol (1:1, v/v). The eluted fraction was reapplied and washed with 3 mL of H2O. Sulfatides were eluted with 2 mL of methanol and 2.5 mL of chloroform:methanol (1:1, v/v). Each fraction was evaporated and suspended in chloroform:methanol (2:1, v/v). Ceramides, cerebrosides and sphingomyelin were quantified by determination of sphingosine with fluorescamine according to the method of Naoi et al. (30) and sulfatides with azure A according to the method of Kean (31).

Statistical analysis.

All data were expressed as means ± sem The effect of diet on VK concentrations in each brain region was analyzed by one-way ANOVA using Sigmastat version 2.0 software (SPSS, Chicago, IL). Differences among the brain regions in each diet group were analyzed by Kruskal-Wallis one-way ANOVA. The effect of diet on sphingolipid concentrations in the brain regions was analyzed by two-way ANOVA. Differences were considered significant at P < 0.05, and a Student-Newman-Keuls posttest was then applied.

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RESULTS

Phylloquinone and MK-4 concentrations.

Phylloquinone and MK-4 concentrations were present in all brain regions, although MK-4 was by far the major form of the vitamin (Table 1). Diet markedly affected K1 and MK-4 concentrations in the various brain regions (P < 0.001), values increasing with K1 intake. Menaquinone-4 was unevenly distributed in the brain (P < 0.01), with differences being particularly marked in rats fed the L and A diets. In these groups, MK-4 concentrations were highest in the midbrain and pons medulla, two regions rich in white matter; in the L group, concentrations in these two regions were markedly higher than in all other regions, whereas in the A group, they were markedly higher than in the cerebellum and striatum (P < 0.05). In the L group, MK-4 concentrations were lower in the frontal cortex and hypothalamus and lowest in the cerebellum, the olfactory bulb, the thalamus, the hippocampus and the striatum. In rats fed the H diet, differences among regions were less marked, consisting only of lower MK-4 concentrations in the hippocampus and striatum (P < 0.05). In contrast to MK-4 distribution, K1 distribution was more homogeneous among brain regions; in the A group, K1 concentration was markedly lower in the cerebellum, whereas in the H group, it was markedly lower in the cerebellum, pons medulla and midbrain. In the L group, K1 concentration did not differ among brain regions.

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TABLE 1

Phylloquinone (K1) and menaquinone-4 (MK-4) concentrations in brain regions of rats fed diets containing low, adequate or high levels of phylloquinone1

Sphingolipid concentrations.

Individual sphingolipid concentrations varied among brain regions (P < 0.001) but were not affected by K1 intake (Table 2). Cerebroside, sphingomyelin and sulfatide concentrations were highest in the pons medulla and the midbrain, two regions rich in white matter, although cerebroside concentrations were also high in the striatum. The cerebellum and frontal cortex generally had the lowest concentrations of cerebrosides, sphingomyelin and sulfatides, whereas the olfactory bulb, the hypothalamus, the thalamus and the hippocampus had intermediate concentrations. Ceramide concentrations were highest in the striatum (P < 0.05) and lowest in the pons medulla, the midbrain and the cerebellum. Ganglioside concentrations were highest and lowest in the frontal cortex and the pons medulla, respectively (P < 0.05), with intermediate concentrations in the other regions.

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TABLE 2

Sphingolipid concentrations in brain regions of rats fed diets containing low (L), adequate (A) or high (H) levels of phylloquinone1

Relationships between MK-4 and sphingolipids in brain.

The specific MK-4–sphingolipid relationship was investigated for each sphingolipid as a function of dietary intake. Correlations were established for each diet group (5 rats/group) considering individual MK-4 and sphingolipid concentrations in all nine brain regions studied. Menaquinone-4 was positively correlated with sulfatides and sphingomyelin, and negatively correlated with gangliosides, with the strength of the relationships decreasing as a function of K1 intake. Correlations between MK-4 and sulfatides (Fig. 1) and between MK-4 and sphingomyelin (Fig. 2) were significant in rats fed the L (MK-4 and sulfatides: r = 0.518, P < 0.001; MK-4 and sphingomyelin: r = 0.515, P < 0.001) and A diets (MK-4 and sulfatides: r = 0.479, P < 0.001; MK-4 and sphingomyelin: r = 0.426, P < 0.001) but not in rats fed the H diet (MK-4 and sulfatides, r = 0.08; MK-4 and sphingomyelin, r = 0.193). Menaquinone-4 was negatively correlated with gangliosides (Fig. 3) in the L (r = −0.398, P < 0.001) and A groups (r = −0.353, P < 0.05) but not the H group (r = 0.099). Correlations between MK-4 and cerebrosides (L, r = 0.226; A, r = 0.259; H, r = 0.177) and MK-4 and ceramides (L, r = −0.284; A, r = −0.245; H, r = −0.125) were not significant.

FIGURE 1
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FIGURE 1

Relationship between brain MK-4 and sulfatide concentrations in rats fed diets containing low (L), adequate (A) and high (H) levels of phylloquinone (n = 5 rats/group). Data points are concentrations from the 9 brain regions of the 5 rats in each diet group.

FIGURE 2
View larger version:
FIGURE 2

Relationship between brain MK-4 and sphingomyelin concentrations in rats fed diets containing low (L), adequate (A) and high (H) levels of phylloquinone (n = 5 rats/group). Data points are concentrations from the 9 brain regions of the 5 rats in each diet group.

FIGURE 3
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FIGURE 3

Relationship between brain MK-4 and ganglioside concentrations in rats fed diets containing low (L), adequate (A) and high (H) levels of phylloquinone (n = 5 rats/group). Data points are concentrations concentrations from the 9 brain regions of the 5 rats in each diet group.

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DISCUSSION

This study was designed to characterize the distribution of vitamin K and sphingolipids in various brain regions and to investigate their regional interactions. To our knowledge, this is the first study to report on K1 and MK-4 concentrations among different anatomical brain entities. As reported for brain homogenates (18,21), VK in the different brain regions was mainly present in the form of MK-4, with this vitamer accounting for >98% of total VK. Brain MK-4 concentrations also increased with K1 intake; overall, MK-4 concentrations for group H were ∼3× and ∼8.2× higher than those for groups A and L, respectively. The presence of MK-4 as the principal form of VK in the various brain regions although K1 was the form added to the diets suggests that dietary K1 is bioconverted into MK-4. Numerous studies report the presence of MK-4 in extrahepatic organs (18,21), and studies by Davidson et al. (22) and Ronden et al. (32) provide evidence that tissue MK-4 is derived by de novo synthesis from K1 in a metabolic process that does not involve the bacterial transformation of intermediate compounds.

The presence of MK-4 at the tissue level and its synthesis from K1 is intriguing and remains largely unexplained. The present study indicates that when dietary K1 intake is low or adequate, MK-4 preferentially accumulates in highly myelinated brain regions. The pons medulla and the midbrain, two regions with high MK-4 concentrations, were also the richest in sphingomyelin, cerebrosides and sulfatides, principal markers of the myelin sheath. It should be mentioned that the sphingolipid distribution reported here agrees with previously published, albeit less detailed, profiles in humans (33), bovines (34) and rats (35).

The strong associations between MK-4 and sphingomyelin and between MK-4 and sulfatides are in accordance with a role for vitamin K in the metabolism of these complex lipids. In a series of experiments, Sundaram et al. (12,13) demonstrated that VK status can alter brain concentrations of sphingolipids, especially sulfatides. Treatment of 16-d-old mice with warfarin for 2 wk decreased brain concentrations of sulfatides, sphingomyelin and cerebrosides by 42, 17 and 12% respectively, but did not affect ganglioside concentration (12). Treatment with warfarin also decreased the activity of brain GST, the key enzyme of sulfatide synthesis, by 19% (13). Feeding the mice an excess of K1 (1 mg/d for 3 d) restored sulfatide concentrations and GST activity but did not affect concentrations of sphingomyelin and cerebrosides, which continued to decline, suggesting that following a period of inhibited synthesis (i.e., warfarin treatment), sulfatide synthesis is prioritized (12).

In the present study and in contrast to Sundaram’s results (12,14), K1 intake did not alter sulfatide concentrations in any of the brain regions. There are several possible explanations for this discrepancy. First, the dietary intervention used in the present study was not as drastic as that used in Sundaram’s studies, which investigated the effect of VK on sphingolipids in extreme circumstances (i.e., severe deficiency and pharmacological supplementation). Second, sulfatides are replaced quite slowly in the adult brain; the estimated half-life of myelin sulfatides is ∼120 d (36). In light of this, the experimental period of the present study may not have been long enough to cause significant changes. Third, brain sulfatide concentrations appear to be highly regulated (37,38). When control 16-d-old mice were treated with K1, the increase in brain sulfotransferase activity was offset by higher activity of arylsulfatase, the principal sulfatide-degrading enzyme; thus, there was no change in sulfatide levels (13). The present study tends to support similar strict biosynthetic control of sulfatide concentrations in rats up to 6 mo of age.

However, the fact that dietary K1 as investigated in the present study did not affect sphingolipid concentrations in various brain regions in no way undermines the finding that when K1 intake is low or adequate, i.e., comparable to what has been observed in the general population (39), MK-4 preferentially accumulates in regions rich in myelin. In addition to the myelination process, the strong negative correlation between MK-4 and gangliosides tends to support a modulatory role for this vitamer in the general sphingolipid biosynthetic pathway.

Studies of cell cultures, animal models and certain diseases suggest important roles for sphingolipids in cell proliferation, differentiation and survival. At low concentrations, sphingomyelin and ceramide can stimulate cell proliferation and survival, whereas higher levels can induce cell dysfunction or death (for a review, see Cutler & Mattson 9). Recent studies show that sphingolipids, including sphingomyelin and ceramide, accumulate in several tissues (e.g., brain and liver) during aging (40,41). In addition, recent data link alterations in sphingomyelin metabolism to the pathogenesis of several age-related diseases, including neurodegenerative disorders, cardiovascular disease, diabetes and cancers (9). Studies of Alzheimer’s disease (AD) point to a significant decrease in brain sulfatide concentrations (42,43) and altered ganglioside composition in some brain regions (10,44). A recent report on the very early stages of AD (45) showed that sulfatides were decreased as much as 93% in gray matter and as much as 58% in white matter, whereas ceramides were increased threefold, suggesting that alterations in sphingolipid metabolism may be involved in the pathological process of AD. In light of this finding and the strong associations found in the present study between MK-4 and sphingomyelin, sulfatides and gangliosides, this vitamer may play an important role in brain function and in the aging process.

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Acknowledgments

The authors thank Jacques Canuel, Odile Dallaire and Stéphane Meynard for their help regarding the maintenance of the animals.

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Footnotes

  • ↵1 Supported by the Natural Sciences and Engineering Research Council of Canada.

  • ↵3 Abbreviations used: A, adequate; AD, Alzheimer’s disease; GST, galactocerebroside sulfotransferase; H, high; K1, phylloquinone; K1H2, 2′,3′-dihydrophylloquinone; L, low; MK-4, menaquinone-4; VK, vitamin K.

  • Manuscript received: July 3, 2003.
  • Initial review completed: August 14, 2003.
  • Revision accepted: October 2, 2003.
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  1. J. Nutr.
    vol. 134 no. 1 167-172
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