- Serum protein levelsin CRCs
After screening according to the inclusion criteria and exclusion criteria, 398 cases of CRC patients were included in the retrospective study. Age-matched healthy volunteers from the physical examination center were selected as the control group. The clinical characteristics of subjects are shown inTable 1. There was no significant difference between the CRC patients and healthy volunteers in sex, age, BMI, personal habits including smoking and drinking, chronic diseases including diabetes and hypertension, and some serum indicators including hemoglobin, alanine transaminase (ALT), glutamic oxaloacetic transaminase (GOT), creatinine, and triglyceride. Serum nutrition-related indicators including total protein and albumin in CRC group were lower than those in control group (p＜0.05). Other serum biochemical indicators such as high density lipoprotein (HDL), low density lipoprotein (LDL), and Apolipoprotein A (apoA) were also statistically different between these two groups.
Clinically, serum albumin level is an objective index for nutritional assessment. HDL, LDL and Apo A are not used as an indicator of nutritional assessment. We further analyzed the nutrition-related indicators including total protein, albumin, and globulin, and the ratio of albumin and globulin in CRC patients in terms of gender, pathological stage, and cancer site. As shown in Table 2, there was no significant difference in the serum nutrition-related indicators at different pathological stages and between males and females. There was no significant difference in the globulin levels and in the ratio of albumin and globulin between the colon cancers and rectal cancers, but the total protein and albumin in colon cancer was lower than that in rectal cancer (p＜0.05).
- Association of gut microbes with different serum albumin levels in CRCs
A total of 30 cases of CRC patients were included to detect the gut microbiota. These patients were divided into three groups according to albumin levels. F0, F1, and F2 represent ranges of albumin values of over 40 g/L, 35~40 g/L and below 35 g/L, respectively. As shown in Table 3, there was no significant difference among these three groups in sex, age, BMI, and some serum indicators including hemoglobin, ALT, GOT, creatinine, triglyceride, HDL, LDL, and apolA.
The gut bacteria were detected to compare the diversity among the three groups. As shown in Supplement material-Figure S2, panels A, B, and C show the Chao1 curves, Shannon curves, and Simpson curves, respectively. There is no statistical difference (Kruskal test) between Chao1 indexes, Shannon indexes, and Simpson indexes among the three groups.
The community structure of gut bacteria among the three groups was analyzed. Figure 1 shows the plot of community structure among the three groups. The stacked bar graph in Panel A of figure 1 shows the relative abundance of gut bacteria at the genus level for each sample. Despite the large variation of the bacteria in each sample, the bacteria that made up the majority of stool samples in CRC patients, in turn, included Bacteroides, Escherichia/Shigella, Prevotella, Ruminococcaceae, Faecalibacterium, Streptococcus, Roseburia, Parabacteroides, Lachnospiraceae, and Clostridium XlVa. The taxonomic tree heatmap in Panel B of figure 1 shows the composition and proportion of microorganisms at different taxonomic levels including phylum, class, order, family, and genus. The outermost layer indicates annotated genus. The orange circles indicate that the abundance of Sutterella (phylum__Proteobacteria, class__Betaproteobacteria, order__Burkholderiales, family__Sutterellaceae, genus__sutterella) is higher in the CRCs with the lower serum albumin level (Kruskal test). The enterotype map inPanel C of figure 1 represents the correlation and contribution of the bacteria to different groups. Orange area, cyan area, and purple area represents F0 group, F1 group, and F2 group, respectively. The result shows that Burkholderia, Grenulicatella, Stenotrophomonas, Collinsella, Actinomyces, Oribacterium, Delftia, and Parasutterella are closely related to F0 group. Ruminococcus, Coprococcus, and Paraprevotella are closely related to F1 group. Prevotella, f_Porphyromonadaceae, Alloprevotella, Methanobrevibacter, Porphyromonas, Coprobacter, Eggerthia, Lactobacillus, Turicibacter, Pseudoflavonifractor, Campylobacter, Sutterella, and Oscilfibacter are closely related to F2 group (Monte-Carlo simulation).
The gut fungus were detected to compare the diversity among the three groups. The Supplement material-Figure S3 shows the alpha diversity analysis of gut fungus among the three groups including F0, F1, and F2. Panels A, B, and C shows the Chao1 curves, Shannon curves, and Simpson curves, respectively. There is no statistical difference (Kruskal test) between Chao1 indexes, Shannon indexes, and Simpson indexes among the three groups.
Figure 2 shows the community structure of gut fungus among the three groups. The stacked bar graph in Panel A of figure 2 shows the relative abundance of gut fungus from the stool samples of CRC patients at the genus level. The fungus that can be successfully compared by the relative database in the top 10 include Saccharomyces, Saccharomycetales, Humicola, Candida, Rhodotorula, Ascomycota, Penicillium, Malassezia, Filobasidium, and Debaryomyces. The taxonomic tree heatmap in Panel B of figure 2 shows that there were statistically significant differences in the abundance of fungus including Agaricomycetes (p__Basidiomycota, c__Agaricomycetes, o__Agaricomycetes_unidentified, f__Agaricomycetes_unidentified_1, g__Agaricomycetes), Simplicillium (p__Ascomycota, c__Sordariomycetes, o__Hypocreales, f__Cordycipitaceae, g__Simplicillium), Sclerotiniaceae (p__Ascomycota, c__Leotiomycetes, o__Helotiales, f__Sclerotiniaceae, g__Sclerotiniaceae), and Exophiala (p__Ascomycota, c__Eurotiomycetes, o__Chaetothyriales, f__Herpotrichiellaceae, g__Exophiala) among the three groups (Kruskal test). The enterotype map inPanel C of figure 2 represents the correlation and contribution of the gut fungus to different groups. Orange area, cyan area and purple area represent F0 group, F1 group, and F2 group, respectively. The result shows that Simplicillium, Bionectria, Verticillium, Hannaella, Davidiella, Thanatephorus, Acrostalagmus, Devriesia, Malassezia, Cyberlindnera, Trichocomaceae, and Asterotremella are closely related to F0group. Eurotiomycetes and Aspergillus are closely related to F1 group. Bionectraceae, Magnusiomyces, Sarocladium, Phoma. Kodamaea, Pichia, Pseudogymnoascus, Incertae_sedis, Trichosporon, Geotrichum, Penicillium, Auricularia, and Pleosporaceae are closely related to F2 group (Monte-Carlo simulation).
- Association of gut microbial metabolites with different serum albumin levels in CRCs
As shown in Table 4, there was no significant difference among these three groups with respect to sex, age, BMI, and some serum indicators including hemoglobin, ALT, GOT, creatinine, triglyceride, HDL, LDL, and apolA. A total of 124 microbial metabolites in 10 classes extracted from stool samples of CRC patients were quantified in the present study. The list of microbial metabolites is showed in Supplement material-table S1.
Figure 3 shows the relative proportion of gut microbial metabolites in each example. The stacked bar on the left represents each sample, and the stacked bar on the right represents a set of the same group. Panel A in Figure 3 shows the proportion of gut microbial metabolites in the detected samples at class level. The results show that the gut microbial metabolites accounting for the top three include amino acids, cinmic acids, and fatty acids at class level. Panel B in Figure 3shows the proportion of gut microbial metabolites in the absolute quantitative top 30. The gut microbial metabolites from CRC patients in the top 10 including acetic acid, L-methionine, 3-hydroxybutyric acid, L-glutamic acid, L-lysine, glutathione, glyceric acid, oxoadipic acid, L-homoserine, and gamma-aminobutyric acid. The heatmap in Figure 4 shows that the gut microbial metabolites had statistically significant differences among the three groups (Kruskal test). The panel A and panel B in Figure 4shows the concentration of gut microbial metabolites from each sample and each group, respectively. The results show that the stool samples in CRC patients with the lower serum albumin level had an increase in the concentration of gut microbial metabolites including 2-hydroxybutyric acid, 2-phenylglycine, D-2-hydroxyglutaric acid, glycine, L-asparagine, behenic acid, oxoglutaric acid and succinic acid, and a decrease in the concentration of gut microbial metabolites including L-norleucine, salicyluric acid, isocitric acid, and glycine.