Supplemental feeding of phospholipid-enriched alkyl phospholipid from krill relieves spontaneous atopic dermatitis and strengthens skin intercellular lipid barriers in NC/Nga mice

Figures & data

Table 1. Composition of test lipids (a) and molecular species at the sn-2 position of krill alkylphospholipid (b).

a					b
	Test lipids
	Krill phospholipid	Soybean phospholipid	Fish oil	Soybean oil	sn-2 position	(%)
Phospholipid subclass (%)					18:1	3.9
Alkenylacyl	-	-			18:2	1.9
Alkylacyl	28.8	1.4			20:4	1.7
Diacyl	51.7	95.0			20:5	39.9
Lysophospholipids	19.5	0.9			22:4	0.1
Others	-	2.7			22:5	1.3
Fatty acid (%)					22:6	51.2
14:0	1.0	-	0.2	-
16:0	10.4	13.5	1.4	10.8
16:1	1.9	-	0.4	4.1
18:0	0.5	3.1	3.1	0.4
18:1	13.5	7.8	5.0	24.9
18:2	2.0	68.2	1.6	52.2
18:3	2.4	7.3	1.2	6.5
20:1	-	-	2.5	0.2
20:5	42.0	-	47.0	-
22:5	-	-	0.4	-
22:6	24.6	-	27.1	-
Others	1.7	-	10.0	0.9

Table 2. Composition of test diets.

	Standard diet	Alk diet	FO diet
Ingredient	g/kg
Corn starch	529.5	529.5	529.5
Sucrose	100	100	100
Casein^a	200	200	200
L-Cystine	3	3	3
Soybean oil	70	60	51
Krill phospholipid^b	0	10	0
Fish oil^c	0	0	9
Soybean phospholipid^d	0	0	10
Cellulose^e	50	50	50
Mineral mix^f	35	35	35
Vitamin mix^f	10	10	10
Choline bitartrate	2.5	2.5	2.5
tert-Butylhydroquinone	0.014	0.014	0.014

^aCasein (ALACID; New Zealand Daily Board, Wellington, New Zealand).

^b30% alkyl type phospholipid extracted from krill.

^cMarinolC38 (Loders Croklaan, Wormerveer, the Netherlands) .

^dEpikuron 200 (95% soybean phosphatidylcholine; Lucas Meyer, Hamburg, Germany).

^eJust Fiber (Morimura Bros., Fort Lee, NJ, USA).

^fMineral and vitamin mixtures were prepared according to the AIN93G formulation.

Table 3. Growth parameters and plasma lipids (cholesterol, triglyceride, free fatty acid) in NC/Nga mice fed test diets for 5 weeks (Experiment 2).^a

		AD			One-way ANOVA
	Normal	Control	Alk	FO	P value
Growth parameters
Food intake (g/day)	3.60 ± 0.05b	3.95 ± 0.10ab	3.83 ± 0.11ab	4.03 ± 0.06a	<0.050
Final body weight (g)	34.8 ± 0.50a	26.2 ± 1.40b	26.3 ± 1.60b	25.8 ± 1.60b	<0.001
Spleen weight (g/100g BW)	0.20 ± 0.01b	0.69 ± 0.10a	0.69 ± 0.12a	0.80 ± 0.17a	<0.050
Plasma lipids (mmol/L)
Cholesterol	3.59 ± 0.30a	2.09 ± 0.09b	2.17 ± 0.20b	2.16 ± 0.15b	<0.001
Triglyceride	1.24 ± 0.18	1.53 ± 0.40	1.43 ± 0.34	1.56 ± 0.32	0.919
Free fatty acid	0.14 ± 0.01	0.16 ± 0.01	0.14 ± 0.02	0.16 ± 0.01	0.390

^aValues are means (n = 6–8 mice per group) and their standard errors. Different superscript letters indicate significant differences at P < 0.05. P value between all groups; Alk: alkyl phospholipid; FO: fish oil.

Figure 1. Inflammation scores for NC/Nga mice fed test diets for 3 weeks in Experiment 1. Test diets were standard (for control), Alk, and FO. Values are means (n = 7 mice per group), with their standard errors represented by vertical bars. Different superscript letters indicate significant differences at P < 0.05. (a) Plasma IgE; (b) scratch frequency; (c) TEWL; (d) ear thickness.

Figure 2. Immunoblot analysis of TJ proteins in the dorsal skin in NC/Nga mice fed test diets for 3 weeks in Experiment 1. Test diets were standard (for control), Alk, and FO. Values are means (n = 7 mice per group), with their standard errors represented by vertical bars. Different superscript letters indicate significant differences at P < 0.05.

Figure 3. Inflammation scores for NC/Nga mice fed test diets for 5 weeks in Experiment 2. Test diets were standard diet (for normal and control), Alk, and FO. Normal is non-onset AD, and others are AD induced by infection with M. musculi. Values are means (n = 6–8 mice per group), with their standard errors represented by vertical bars. Different superscript letters indicate significant differences at P < 0.05. (a) Plasma IgE; (b) scratch frequency; (c) TEWL; (d) ear thickness.

Table 4. Plasma plasmalogen-related parameters and total phospholipid concentration in NC/Nga mice fed test diets for 5 weeks (Experiment 2).^a

		AD			One-way ANOVA
	Normal	Control	Alk	FO	P-value
Pls(Cho+Etn) (μmol/L)	19.2 ± 0.8a	11.6 ± 0.6b	13.7 ± 0.9b	11.4 ± 0.9b	<0.001
PL (mmol/L)	4.59 ± 0.27a	3.28 ± 0.15b	3.12 ± 0.34b	3.04 ± 0.10b	<0.001
PlsCho/PL (mol%)	0.16 ± 0.02ab	0.14 ± 0.01b	0.17 ± 0.01a	0.15 ± 0.01ab	0.121
PlsEtn/PL (mol%)	0.27 ± 0.02ab	0.22 ± 0.01b	0.28 ± 0.02a	0.23 ± 0.01b	<0.050
Pls(Cho+Etn)/PL (mol%)	0.42 ± 0.03a	0.35 ± 0.02b	0.46 ± 0.03a	0.38 ± 0.02ab	<0.050

^aValues are means (n = 6–8 mice per group) and their standard errors. Different superscript letters indicate significant differences at P< 0.05 (one-way analysis of variance followed by Tukey-Kramer method). Alk: alkyl phospholipid; FO: fish oil; PL: total phospholipid; PlsCho: choline plasmalogen; PlsEtn: ethanolamine plasmalogen.

Figure 4. Plasmalogen and alkyl phospholipid concentration in plasma of NC/Nga mice fed test diets for 5 weeks in Experiment 2. Test diets were standard diet (for normal and control), Alk, and FO. Normal is non-onset AD, and others are AD induced by infection with M. musculi. Values are means (n = 6–8 mice per group), with their standard errors represented by vertical bars. Different superscript letters indicate significant differences at P < 0.05. AlkCho: choline alkylphospholipids; AlkEtn: ethanolamine alkylphospholipids; PlsEtn: ethanolamine plasmalogen; PlsCho: choline plasmalogen.

Figure 5. Distribution of molecular species at the sn-2 position of (a) choline alkylphospholipid (AlkCho), (b) ethanolamine alkylphospholipid (AlkEtn), (c) ethanolamine plasmalogen (PlsEtn), and (d) choline plasmalogen (PlsCho) in the dorsal skin of NC/Nga mice fed test diets for 5 weeks in Experiment 2. Test diets were standard diet (for normal and control), Alk, and FO. Normal is non-onset AD, and others are AD induced by infection with M. musculi. Values represent skin concentrations of FAs at the sn-2 position of individual ether glycerophospholipids and are means (n = 6–8 mice per group), with their standard errors represented by vertical bars. Different superscript letters indicate significant differences at P < 0.05.

Figure 6. Total phospholipid concentration in the dorsal skin of NC/Nga mice fed test diets for 5 weeks in Experiment 2. Test diets were standard diet (for normal and control), Alk, and FO. Normal is non-onset AD, and others are AD induced by infection with M. musculi. Values are means (n = 6–8 mice per group), with their standard errors represented by vertical bars. Different superscript letters indicate significant differences at P < 0.05.

Figure 7. Levels of intercellular lipid in the dorsal skin of NC/Nga mice fed test diets for 5 weeks in Experiment 2. Test diets were standard diet (for normal and control), Alk, and FO. Normal is non-onset AD, and others are AD induced by infection with M. musculi. Values are means (n = 6–8 mice per group), with their standard errors represented by vertical bars. Different superscript letters indicate significant differences at P< 0.05. (a) Total cholesterol; (b) FFA; (c) non-hydroxyceramide; (d) hydroxyceramide.

Figure 8. The relationship between levels of skin hydroxyceramide and total plasmalogens (a), total alkyl phospholipid (b), and total phospholipid (c) in the plasma of NC/Nga mice fed test diets for 5 weeks in Experiment 2. Values (r) represent Pearson’s product–moment correlation coefficient.