A Phospholipid-Protein Complex from Antarctic Krill Reduced Plasma Homocysteine Levels and Increased Plasma Trimethylamine-N-Oxide (TMAO) and Carnitine Levels in Male Wistar Rats

Abstract

Seafood is assumed to be beneficial for cardiovascular health, mainly based on plasma lipid lowering and anti-inflammatory effects of n-3 polyunsaturated fatty acids. However, other plasma risk factors linked to cardiovascular disease are less studied. This study aimed to penetrate the effect of a phospholipid-protein complex (PPC) from Antarctic krill on one-carbon metabolism and production of trimethylamine-N-oxide (TMAO) in rats. Male Wistar rats were fed isoenergetic control, 6%, or 11% PPC diets for four weeks. Rats fed PPC had reduced total homocysteine plasma level and increased levels of choline, dimethylglycine and cysteine, whereas the plasma level of methionine was unchanged compared to control. PPC feeding increased the plasma level of TMAO, carnitine, its precursors trimethyllysine and γ-butyrobetaine. There was a close correlation between plasma TMAO and carnitine, trimethyllysine, and γ-butyrobetaine, but not between TMAO and choline. The present data suggest that PPC has a homocysteine lowering effect and is associated with altered plasma concentrations of metabolites related to one-carbon metabolism and B-vitamin status in rats. Moreover, the present study reveals a non-obligatory role of gut microbiota in the increased plasma TMAO level as it can be explained by the PPC's content of TMAO. The increased level of carnitine and carnitine precursors is interpreted to reflect increased carnitine biosynthesis.

Keywords: Euphausia superba; Omega-3 polyunsaturated fatty acids; carnitine; homocysteine; one-carbon metabolism; phospholipid-protein complex; trimethylamine-N-oxide.

Figure 1

Metabolites of the folate cycle, transsulfuration pathway, and methionine (Met)-homocysteine (Hcy) cycle. Enzymes are shown in grey boxes, and their vitamin co-factors in green circles. Abbreviations: PC, phosphatidylcholine; MetSO, methionine sulfoxide, TMA, trimethylamine; TMAO, trimethylamine-N-oxide; FMO, flavin-containing monooxygenase; BHMT, betaine homocysteine methyltransferase; DMG, dimethylglycine; CTH, cystathionine gamma lyase; CBS, cystathionine beta-synthase; MS, methionine synthase/5-methyltetrahydrofolate-homocysteine methyltransferase (MTR); THF, tetrahydrofolate; 5-mTHF, 5-methyltetrahydrofolate; 5,10-mTHF, 5,10-methylenetetrahydrofolate; MTHFR, methylenetetrahydrofolate reductase (NAD(P)H); MSR, methionine synthase reductase.

Figure 2

Plasma levels of metabolites of the transsulfuration and homocysteine remethylation pathways. Rats were fed either a control diet (2% soy oil, 8% lard, 20% casein), or an experimental diet where casein and lard were replaced with phospholipid-protein complex (PPC) at 6% or 11% (wt%) for 4 weeks. Total homocysteine (tHcy; A), methionine (Met; B), Met sulfoxide (C), cysteine (D), dimethylglycine (DMG; E), and betaine (F) were measured in fasting plasma samples. Values shown are means with standard deviation (n = 6). One-way analysis of variance (ANOVA) with Dunnet’s post hoc test was used to determine values significantly different from control (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 3

Plasma levels of B-vitamins and derivatives. Male Wistar rats were fed either a control diet (2% soy oil, 8% lard, 20% casein), or an experimental diet where casein and lard were replaced with phospholipid-protein complex (PPC) at 6% or 11% (wt. %) for 4 weeks. Quinolinic acid (vitamin B3, A), pyridoxal phosphate (PLP; vitamin B6, B), cobalamin (vitamin B12, C), and 5-methyltetrahydrofolate (5-mTHF; D) were measured in fasting plasma samples. Values shown are means with standard deviation (n = 6). One-way analysis of variance (ANOVA) with Dunnet’s post hoc test was used to determine values significantly different from control (* p < 0.05).

Figure 4

Plasma levels of phosphatidylcholine (PC) degradation products and carnitine precursors. Male Wistar rats were fed either a control diet (2% soy oil, 8% lard, 20% casein), or an experimental diet where casein and lard were replaced with phospholipid-protein complex (PPC) at 6% or 11% (wt%) for four weeks. Choline (A), trimethylamine-N-oxide (TMAO; B), carnitine (C), γ-butyrobetaine (D) and trimethyllysine (TML; E) were measured in fasting plasma samples. Values shown are means with standard deviation (n = 6). One-way analysis of variance (ANOVA) with Dunnet’s post hoc test was used to determine values significantly different from control (** p < 0.01, *** p < 0.001).

Figure 5

Correlation between trimethylamine-N-oxide (TMAO) and intermediates in TMAO synthesis. Plasma levels of TMAO and trimethyllysine (A), TMAO and carnitine (B), TMAO and γ-butyrobetaine (C), and TMAO and choline (D), were analyzed by linear regression. Data were obtained as described under legend to Figure 4. Control group is depicted in green, 6% phospholipid-protein complex (PPC) in blue, and 11% PPC in red.