3. Discussion
This is the first study, using a randomized controlled cross-over feeding trial, to compare the effect of diets differing in the primary source of protein, AP or SP, on plasma metabolite profiles. Overall, based on the VIP score of each metabolite, the major differences were observed for some metabolites in the classes of PE and were amino acid-related. The scores were higher after the participants consumed the AP rather than the SP diet. Consistent with the constituent component of soybeans, ornithine-related and isoflavones metabolites were higher after the participants consumed the SP rather than the AP diet. A potentially favorable clinical biomarker identified after the participants consumed the SP diet was plasma PE O-38:6 (PE O-18:1_20:5), which was negatively correlated with the plasma TG and VLDL-C concentrations and positively correlated with plasma HDL-C and apoA1 concentrations.
Prior reports have identified some metabolite biomarkers for different types of dietary protein [
14,
15]. The European Prospective Investigation into Cancer and Nutrition (EPIC) study identified 3-methylhistidine as a potential biomarker for dietary poultry and some fish species [
9]. Three-methylhistidine is stored in skeletal muscle. Carnosine, a dipeptide also present at high concentrations in muscle tissue, is composed of β-alanine and histidine. Our finding of higher concentrations of 3-methylhistidine and β-alanine after the participants consumed the AP compared to SP diet, during a controlled dietary feeding protocol, is consistent with that observed in two observational studies reporting positive correlations between these two amino acid-related metabolites and meat intake [
11,
14], in addition to EPIC. Our study design precluded an assessment of the biomarkers that distinguished among red meat, poultry or fish consumption. However, the network analysis indicated that the pathways for β-alanine, histidine and 3-methylhistidine metabolism were all significantly upregulated in the participants at the end of the AP relative to the SP diet phase, supporting the potential value of these amino acids as biomarkers for protein type.
In the current study, lipidomic analyses indicated that the majority of the metabolites that differed in response to the two diets belonged to the phospholipid class. Three of them, PE O-37:5 (PE O-17:1_20:4), PE P-36:5 (or PE O-36:6) and PE O-38:6 (PE O-18:1_20:5), were significantly higher after the participants consumed the AP compared to the SP diet. Similar differences between diets high in animal compared to plant proteins have been reported in one [
15], but not other, observational studies [
16,
17,
18]. The relations between individual phospholipid species and dietary protein type are complex, and the disparities observed among studies may be due to differences in the platforms used to measure the metabolites, methods used to assess dietary protein type and specific types of animal and plant proteins consumed [
11,
15,
16,
17,
18].
The plasma concentrations of N-α-acetyl-L-ornithine and 3-aminotyrosine were significantly higher after the participants consumed the SP compared to the AP diet. From a protein quality perspective, SP is complete in terms of its amino acid profile, and particularly rich in phenylalanine, glutamate and arginine. These three amino acids are precursors of ornithine, and thus provide substrates for the urea cycle, arginine synthesis and aromatic amino acid metabolism. It has previously been reported that the L-ornithine supplement activated the urea cycle by increasing ornithine, necessary to transport amino groups out of mitochondria to the cytoplasm for subsequent urinary excretion [
19]. These data suggest that N-α-acetyl-L-ornithine may be a plasma biomarker for dietary SP. However, this premise is tempered because 3-aminotyrosine is an intermediate in aromatic amino acid metabolism and can be formed via tyrosine metabolism. It has been proposed that phenylalanine is converted to tyrosine and involved in tyrosine metabolism or tyrosine modification [
20].
The plasma concentrations of isoflavones, daidzein 4-sulfate, daidzein and genistein were all significantly higher after the participants consumed the SP compared to the AP diet. These findings, documented previously, reflect the abundance of these compounds in SP [
21,
22,
23]. The SP diet contained 15.4 mg of daidzein and 47.9 mg genistein per 1000 kcal, whereas the AP diet contained 0.6 mg of daidzein and 9.8 mg genistein per 1000 kcal [
13]. However, the microbiome can modify the isoflavones prior to absorption, hence making the levels somewhat tenuous [
24]. Hence, further studies are warranted to determine whether isoflavones are appropriate plasma biomarkers for soybean consumption.
Of the PE metabolites identified that differed between the AP and SP diets, PE O-38:6 (PE O-18:1_20:5) was the most closely associated with CMRFs. PEs have many cellular functions, such as lipid homeostasis, serving substrates for post-translational modification [
25]. More specifically, they play a crucial role in VLDL-C assembly and secretion, and the compositions of PEs and other phospholipids in nascent plasma VLDL-C are highly associated with that in the liver [
25,
26,
27]. Additionally, once released into circulation, PEs are rapidly removed from VLDL-C as the particles are delipidated [
26]. In contrast to prior reports, the present study plasma PE O-38:6 (PE O-18:1_20:5) was negatively associated with VLDL-C concentrations [
25,
26,
27]. This discrepancy may be due to differences in the blood fraction of the PEs that was measured, plasma as in the current study or VLDL-C particles. Data from a global CTP:phosphoethanolamine cytidylyltransferase knockout mouse model (Pcyt2+/−) indicate that a lack of PE globally did not alter hepatic VLDL-C secretion. The plasma VLDL-C concentrations in Pcyt2+/− mice were higher compared to those in wide-type mice [
27]. In contrast, hepatic Pcyt2+/− mice developed steatosis and had lower hepatic VLDL-C secretion [
25]. This suggests that only hepatic PEs may have a positive association with plasma VLDL-C concentration, and PEs in circulation or from other tissues may have a negative association with VLDL-C.
The associations observed between the plasma PEs and CMRFs after the participants consumed the SP diet suggest potential involvement in cholesterol, TG and lipoprotein metabolism, particularly for PE O-38:6 (PE O-18:1_20:5). Previous studies indicated that plasma omega-3 fatty acids have been positively associated with increased HDL-C, lowered plasma TG, VLDL-C and apolipoprotein B [
28,
29]. Given the relatively high in docosahexaenoic acid and eicosapentaenoic acid (EPA)-content in PE O-38:6 (PE O-18:1_20:5) in the participants consuming the SP diet, we postulate there is a synergistic effect of omega-3 fatty acids, and this PE species is resulting in this observation.
A strength of this study is that the plasma samples and CMRF data were collected after the participants consumed both the AP and SP diets in a random order as part of a controlled cross-over trial. The diets were carefully designed to differ primarily in the type of protein and were matched for fiber, cholesterol, macronutrient distribution and fatty acid profile. The cross-over design minimized confounding due to inter-individual differences. A wide range of metabolites were measured by using a combination of three analytical platforms. As expected, the plasma isoflavone concentrations were the primary biomarkers identified after the consumption of the SP and confirm a high level of compliance with the study protocol by the study participants. A limitation of the study is that the AP diet contained a mixture of animal proteins (red meat, chicken, eggs and dairy products); hence, the effect of individual protein sources could not be determined. Although the samples were stored at −80 °C and never thawed, they were collected approximately 15 years ago; thus, we cannot rule out potential metabolite loss or degradation. Only the identified metabolites were assessed. Further research is needed to identify potential unknown metabolites that differed by dietary protein type.