- Email: [email protected]
The serum SA levels are significantly increased in sepsis but decreased in cirrhosis
CHAPTER TWENTY
The serum SA levels are significantly increased in sepsis but decreased in cirrhosis Xiaodan Huanga,b, Qin Yaoc, Lijuan Zhanga,*, Zibin Tianb,*
a Systems Biology and Medicine Center for Complex Diseases, Affiliated Hospital of Qingdao University, Qingdao, China b Department of Internal Medicine, Affiliated Hospital of Qingdao University, Qingdao, China c Department of Gynecology, Affiliated Hospital of Qingdao University, Qingdao, China *Corresponding authors: e-mail address: [email protected]; [email protected]
Contents 1. Introduction 2. Serum/plasma SA concentration measurement 3. Serum SA levels in 64 different types of diseases 3.1 Methods 3.2 Results 4. Discussions and conclusions Acknowledgment Conflict of interest References
336 338 339 339 339 343 345 345 346
Abstract Most of proteins in human blood circulation are glycoproteins with one or more covalently linked N- or O-linked glycans. Sialic acid (SA) generally occurs as the terminal monosaccharide on the glycans. SA in glycoproteins modulates a wide range of physiological and pathological processes and has been routinely measured in hospital since 1950s. Increased serum SA levels have been associated with different types of cancers. However, a systematic comparison of the serum SA levels in different types of human diseases has not been reported. In current study, 160,537 clinical lab test results of serum SA levels from healthy individuals and patients with 64 different types of diseases during the past 5 years in our hospital were retrieved and analyzed. Based on the mean (SD), median, and p (Log10 p) values, we found that patients suffering 55 different types of cancer and noncancer diseases such as sepsis, pancreatitis, bone cancer, rheumatoid arthritis, pancreatic cancer, and encephalitis had significantly (p < 0.05, Log10 p > 1.30) increased median serum SA levels whereas patients suffering hepatic encephalopathy, cirrhosis, renal cyst, and hepatitis had significantly decreased median serum SA levels compared to that of healthy controls. Moreover, the greatest increase in the mean (SD) and Log10 p values was observed in sepsis and pancreatitis, respectively,
Progress in Molecular Biology and Translational Science, Volume 162 ISSN 1877-1173 https://doi.org/10.1016/bs.pmbts.2019.01.009
#
2019 Elsevier Inc. All rights reserved.
335
336
Xiaodan Huang et al.
but not in cancers. Thus, the regulations of serum SA levels were much more complicated than previously assumed. Understanding the molecular mechanisms behind these observations would make serum SA a useful biomarker to facilitate personalized diagnosis and treatment for patients with different diseases.
1. Introduction More than 70% of human proteins are glycoproteins,1,2 which makes glycosylation the most common protein posttranslational modifications. N-linked and O-linked glycans are two most common types of glycans and are ubiquitous in all biological systems. Sialic acids (SAs) are a diverse family of naturally occurring 2-keto-3-deoxy-nonoinc acids with over 50 members. The most abundant SA in human is N-acetylneuraminic acid (Neu5Ac), which is one of the 10 monosaccharides found in N- or O-linked glycan chains. SA biosynthesis begins with conversion from UDP-N-acetylglucosamine (UDP-GlcNAc) by UDP-GlcNAc 2-epimerase to N-acetylmannosamine (ManNAc). CMP-Neu5Ac produced from ManNAc is the high energy donor for sialyltransferases that install SA to the terminal of the N- or O-linked glycan chains. In humans, 20 sialyltransferases transfer SA to other monosaccharides such as galactose (Ga1), N-acetylgalactosamine (N-GalNAc), GlcNAc, or other SA on the glycan chains with α-2,3, α-2,6, α-2,8, or α-2,9 linkage.3 Since no other monosaccharides can add to SA except that SA can add to itself, SA is the outmost monosaccharide of both N-linked and O-linked glycan chains.4 Being particularly prominent on cell surface and secreted molecules, for example, up to 10 million SAs per human erythrocyte and 100 mM estimated SA local concentrations on cell surface,5 SA-containing glycan chains are important players in many physiological and pathological processes.6,7 The negatively charged SA can mask the glycan chains on cell surface proteins by preventing the attack of glycosidase to maintain the integrity of epithelial cells from harmful substances and pathogens.8 At the same time, SA can help pathogenic microorganisms to escape the immune recognition of host, which happens when the pathogenic microorganisms are covered with SA-containing glycans. Yuki et al.9 and Hughes et al.10 reported such incidents when infected with Campylobacter jejuni, a bacterium containing SA-containing lipo-oligosaccharides on its wall, the patients suffered Guillian– Barre syndrome, an acute systemic peripheral neuropathy of quadriplegia.
The serum SA levels are significantly increased in sepsis
337
SA also plays an important role in several human viral infections since several viruses such as influenza viruses, adenoviruses, and rotaviruses can bind to host SA-containing glycans. More specifically, the influenza viruses have hemagglutinin activity glycoproteins (HA) on their surfaces that bind to specific SA-containing glycans on the surface of human erythrocytes and on the epithelial cell membranes of the upper respiratory tract.11,12 Such binding forms the basis of hemagglutination when the virus is mixed with erythrocytes. Such binding is also required for the entry of the influenza virus into epithelial cells of the upper respiratory tract. Widely used antiinfluenza drugs (oseltamivir and zanamivir) are SA analogs that interfere with release of newly generated viruses from infected cells by inhibiting the influenza viral enzyme neuraminidase.13 In human, the ligands for SA are diverse, such as hormones, lectins, antibodies, and inorganic ions, which mainly regulate the adhesion process of cells and cells in inflammation and immune response.14,15 The high density of SAs in extracellular membrane is important in maintaining normal filtration of the glomerular basement membrane.4 The brain tissue in the human contains more SAs than any other tissues. Poly-sialic acid (PSA) is uniquely present in brain tissues. PSA is a homogeneous polymer consisting of α-2, 8 linkages of SA and attach to the nervous system neuroadhesive molecules mainly through typical N-linked glycans. PSA can stimulate the growth of neuronal axons16,17 and assist nerve cell adhesion molecules (NCAM) complete synaptic effects for synaptic remodeling and stability.18 Gangliosides are a group of glycosphingolipids containing SA19 and composed of ceramide and SA-containing glycan chains. Gangliosides are concentrated in axonal terminals and synaptic terminals to maintain long-term stability of axon-myelin and to inhibit excessive axon regeneration after axonal injury.20 Other studies have found that the presence of large amounts of PSA-NCAM and neuroglycolipids in the hippocampus indicates that SA-containing glycans are closely related to the cognitive function of the hippocampus.21–23 About 60 genes are known to be involved in SA biology, where the GNE is responsible for SA synthesis. Mutations in GNE gene cause an autosomal recessive distal myopathy called conjugated vacuole distal myopathy (DMRV). In these patients, reduced ability to generate SAs leads to the damage of cell network.24 Under the electron microscope, rimmed vacuoles in the muscle fibers, abnormal protein deposition in the cells, and different diameters of the muscle fibers are observed in these patients.25 The clinical feature of DMRV is distal muscle atrophy and weakness. In addition, the mutation of SC17A5 gene can lead to SA storage diseases that are involved
338
Xiaodan Huang et al.
in the defect of transporting SA from lysosomes to cytosol.26,27 These patients usually have severe developmental delay, rough face, hepatosplenomegaly, and increased urinary free SA.27 Increased serum SA levels have been reported in patients with different types of cancers, such as, cholangiocarcinoma,28 cervical cancer,29 colorectal cancer,30 breast cancer,31 oral cancer,32 and other types of cancers.33 The increased serum SA levels are associated with the overexpression or increased activity of sialyltransferase in cancer cells.33 Almaraz et al. reported that by increasing the cellular levels of CMP-Neu5Ac, sialylation is greatly enhanced not in all but a subset of the glycans on specific proteins.34 Tumor cells that shed or secrete SA-containing glycoproteins into blood circulation might be another cause of elevated serum SA levels. However, increased serum SA levels are also reported in noncancer diseases, such as inflammation,35,36 atherosclerosis,37,38 rheumatoid arthritis,39 women during pregnancy,40–42 bacterial infections, and liver diseases.43–45 Since the serum SA levels in patients suffering different cancer or noncancer diseases have not been systematically studied and compared, such information would be needed to understand the molecular nature and the meaning of elevated serum SA levels. Thus, in current study a total of 160,537 clinical lab test results of serum SA levels from healthy individuals and patients with 64 different types of diseases during the past 5 years were retrieved and analyzed.
2. Serum/plasma SA concentration measurement Colorimetric assays are the detection methods for serum SA levels including resorcinol colorimetry and periodic acid/thiobarbiturate methods developed since 1950s.46–48 Between 98% and 99.5% of SA in serum is bound to the N- or O-linked glycans in glycoproteins and the rest is either free or bound to lipids in the form of gangliosides.49 Normal serum SA levels in healthy individuals are in the range of 1.6–2.8 mM while the free SA is in the range of 0.5–3 μM. Even though the colorimetric assays are not very sensitive, these assays are sufficient in measuring serum SA levels due to the mM ranges of SA in healthy individuals and increased levels in most of patients. With the rapid development of clinical laboratory techniques, many assays with increased accuracy and sensitivity have been developed. Enzymatic assay is one of the assays that can avoid the disadvantages of low sensitivity and many interfering factors compared with the colorimetric method. Teshima et al.50 and Hoviuchi et al.51 further improved the
The serum SA levels are significantly increased in sepsis
339
traditional enzymatic method to reduce the interference of pyruvic acid on SA measurement. Chromatographic methods can separate SA from other interfering compounds and quantitatively measure the serum SA levels. The high performance anion chromatography-pulse amperometric detection (HPAEC–PAD),52 high performance liquid chromatographic (HPLC),53 gas chromatography with mass spectrometry (GC–MS),54 and liquid chromatography coupled mass spectrum method (LC–MS)55,56 are some of the currently used chromatographic methods.55–58
3. Serum SA levels in 64 different types of diseases 3.1 Methods We have collected the lab data of serum SA levels of both healthy individuals and patients with clinically defined various diseases from the clinical laboratory of Affiliated Hospital of Qingdao University during the past 5 years. Each type of disease that has more than 30 independent test results for serum SA levels was included in current study. As a result, a total of 160,537 clinical lab results of SA from 64 different types of diseases were used in current study. Statistical analysis was performed by using SPSS version 19. Due to nonnormal distribution, Mann–Whitney test was used for statistical analysis. To compare the differences in p values among different types of diseases with that of healthy control, Log10 p values were calculated and used. Twosided test with a 5% significant level (p < 0.05 or Log10 p > 1.30) was considered as statistically significant.
3.2 Results The p value is used in the context of null hypothesis testing in order to quantify the idea of statistical significance of evidence. In essence, the statistical significance is assumed valid when the p values are far apart from that of control values. Based on the data collected, the mean (SD), median, and Log10 p values of serum SA levels for each type of diseases were listed in Table 1. The mean serum SA value is 58.00 U/mL in healthy controls. The data in Table 1 showed that 55/64 diseases in addition to healthy elderlies (>65 years old) had their median values significantly higher than that of healthy controls. In contrast, patients with hepatic encephalopathy, cirrhosis, renal cyst, hepatitis, and bladder stone had their median values lower than that of healthy controls, but for patient with bladder stone, the decreased median values had no statistical significance (Log10 p values
340
Xiaodan Huang et al.
Table 1 The mean, median, and Log10 p values of serum SA levels (U/mL) for healthy controls and patients with cancer and noncancer diseases. 2Log10 SA # of tests Mean (SD) Median p value
Sepsis
278
78.56 (22.21)
79.00
67.41
Pancreatitis
1708
76.46 (15.54)
75.00
>289
Esophagus cancer
3599
74.65 (16.59)
71.70
>289
Bone cancer
108
71.25 (15.30)
71.00
20.15
Rheumatoid arthritis
256
73.00 (14.43)
70.15
71.30
Pancreatic cancer
910
71.41 (13.92)
70.00
168.67
Encephalitis
640
70.22 (14.27)
69.00
98.00
Lung fibrosis
318
70.30 (13.35)
68.35
63.01
Ankylosing spondylitis
69
69.13 (11.09)
68.30
16.24
Brain trauma
539
71.44 (16.95)
68.00
71.19
Lung cancer
8840
71.36 (14.70)
68.00
>289
Intracranial hemorrhage
3345
70.63 (14.33)
68.00
>289
Aplastic anemia
671
70.26 (14.75)
67.50
100.35
Lupus erythematosus
835
68.07 (10.52)
67.50
138.24
Uremia
3785
69.39 (11.65)
67.30
>289
Gout
1366
69.41 (14.34)
67.00
162.52
Ovarian cancer
2019
70.00 (12.93)
66.90
>289
Chronic obstructive PD
1534
67.87 (12.36)
66.00
169.57
Acute myocardial infarction
2490
67.41 (12.52)
65.20
205.04
Azotemia
382
66.88 (11.04)
65.15
50.75
Colon cancer
5534
67.56 (13.56)
65.00
288.86
Diabetic nephropathy
505
66.52 (10.74)
65.00
62.45
Cystitis
49
65.12 (8.05)
65.00
8.03
Preeclampsia
963
65.69 (7.87)
64.80
140.76
Anemia
1739
67.55 (15.17)
64.60
107.60
Nephrotic syndrome
2660
65.34 (9.37)
64.40
216.59
Gastric cancer
11,755
67.66 (15.29)
64.30
286.43
341
The serum SA levels are significantly increased in sepsis
Table 1 The mean, median, and Log10 p values of serum SA levels (U/mL) for healthy controls and patients with cancer and noncancer diseases.—cont’d 2Log10 SA # of tests Mean (SD) Median p value
Endometrial cancer
1035
66.04 (9.77)
64.10
126.63
Gastric ulcer
289
66.78 (14.85)
64.00
17.60
Liver cancer
237
66.03 (13.66)
64.00
14.66
Postbreast surgery
32
67.56 (11.07)
63.95
6.51
Necrosis of femoral head
166
66.26 (11.13)
63.85
20.35
Cervical cancer
1854
65.34 (9.71)
63.80
163.09
Bone fracture
1938
65.44 (11.82)
63.30
111.97
Myeloproliferative disorder
1113
67.03 (16.08)
63.10
48.45
Multiple myeloma
1938
67.08 (18.05)
63.00
66.06
Lymphoma
4305
66.33 (13.52)
63.00
187.33
Rectum cancer
7268
65.82 (13.33)
63.00
194.30
Alzheimer’s disease
95
65.52 (11.61)
63.00
10.32
Nephritis
1532
64.81 (10.87)
63.00
93.77
Psoriasis
98
63.47 (10.09)
62.25
6.32
Asthma
600
63.99 (10.76)
62.00
31.46
Kidney cancer
1282
63.84 (10.48)
61.70
58.75
Acute cerebral infarction
9286
63.50 (10.71)
61.70
151.37
Knee-joint degenerative diseases
432
62.20 (7.83)
61.50
19.35
Bladder cancer
941
63.18 (12.31)
61.00
19.40
Osteoarthritis
74
63.71 (10.02)
60.75
5.00
Breast cancer
3737
61.42 (6.64)
60.70
82.46
Hyperuricemia
118
61.21 (7.24)
60.55
4.04
Leukemia
7627
63.64 (14.29)
60.50
48.74
Coronary heart disease
20,613
62.30 (10.44)
60.50
99.34
Type 2 diabetes mellitus
10,022
61.01 (9.65)
59.40
30.61
Breast lumps
81
60.07 (6.54)
59.30
1.54
Cerebrovascular disease
4560
60.72 (9.54)
59.10
15.27 Continued
342
Xiaodan Huang et al.
Table 1 The mean, median, and Log10 p values of serum SA levels (U/mL) for healthy controls and patients with cancer and noncancer diseases.—cont’d 2Log10 SA # of tests Mean (SD) Median p value
Cerebral arteriosclerosis
751
59.91 (8.21)
59.00
2.64
Osteoporosis
247
59.63 (9.15)
59.00
0.68
Wilms’ tumor
225
61.03 (11.33)
58.50
0.92
Healthy controls >65 years old
531
59.23 (6.35)
58.30
1.74
Cerebral ischemia
2194
59.18 (8.10)
58.10
0.59
Gastritis
2633
58.99 (7.99)
58.00
0.65
Healthy controls
6053
58.61 (6.24)
58.00
0.00
Bladder stone
157
59.93 (10.13)
57.80
0.41
Hepatitis
2831
56.40 (9.54)
56.90
20.86
Renal cyst
496
56.93 (7.64)
56.25
9.32
Cirrhosis
6703
50.33 (12.03)
50.40
>289
Hepatic encephalopathy
77
47.83 (13.08)
45.50
13.44
>1.30). The following diseases had the highest mean values in a descending order: sepsis (78.56), pancreatitis (76.46), esophagus cancer (74.65), rheumatoid arthritis (73.00). The following diseases had the highest SD values: sepsis (22.21), multiple myeloma (18.05), brain trauma (16.95), esophagus cancer (16.59), and myeloproliferative disorder (16.08). To make the data more intuitive, scatter diagram of serum SA levels in the 48 diseases with Log10 p values >15 in comparison to the healthy controls was plotted with lower quartile (25%), median (50%), and upper quartile (75%) marked and showed in Fig. 1. Patients with sepsis had the highest median value followed by patients with pancreatitis, esophagus cancer, bone cancer, rheumatoid arthritis, and pancreatic cancer whereas patients suffering cirrhosis and hepatitis had the lowest median values. The Log10 p values were further plotted and showed in Fig. 2. The highest Log10 p values mean the highest difference in the patients as a group compared to the healthy controls as another group. According to the Log10 p values, the increased serum SA levels worked best as biomarkers for patients with pancreatitis, esophagus cancer, lung cancer, intracranial hemorrhage, uremia, and ovarian cancer among other diseases (Table 1 and Fig. 2).
The serum SA levels are significantly increased in sepsis
343
Fig. 1 Serum SA levels in 48 different types of diseases. The data were sorted in a descending order of the median values. Serum SA levels for each type of disease with lower quartile (25%), median (50%), and upper quartile (75%) ranges were marked in red.
Fig. 2 Log10 p values of SA in 48 different types of diseases compared to that of healthy controls. When the serum SA levels were lower than that of healthy controls, blue color was used in the plot whereas when the serum AFP levels were higher than that of healthy controls, red color was used in the plot.
4. Discussions and conclusions Aberrant glycosylation and sialylation of glycan chains has been recognized as one of the hallmarks of cancers and abnormal immune systems.4,6,7,13,49 Increased serum SA levels have been tested routinely in hospital mainly for the purposes of cancer monitoring and prognosis.33 However, based on data obtained from the serum SA levels from 154,484 patients with 64 clinically defined diseases as well as serum SA levels from 6053 healthy individuals (Table 1), our results showed that: (1) The median serum SA levels in patients with 55 different types of diseases were significantly increased (Log10 p > 1.30) compared to that in healthy controls
344
Xiaodan Huang et al.
(Table 1); (2) Patients with sepsis had the highest mean serum SA levels and the highest SD value among the 64 diseases (Table 1); (3) Based on the Log10 p values, the serum SA levels worked best as biomarkers for patients with pancreatitis, esophagus cancer, lung cancer, intracranial hemorrhage, uremia, ovarian cancer, colon cancer, gastric cancer, nephrotic syndrome, acute myocardial infarction, rectum cancer, and lymphoma among many other cancers and noncancer diseases (Table 1 and Fig. 2); (4) Patients with hepatic encephalopathy, cirrhosis, and hepatitis had both lowest mean and median serum SA levels. Among the three liver diseases, patients with hepatic encephalopathy had the lowest mean but the biggest SD values. Therefore, the increased serum SA levels were not specifically associated with cancers but the decreased serum SA levels were directly associated with liver diseases in the measures of the mean (SD), median, or Log10 p values of our current studies (Table 1 and Figs. 1 and 2); (5) Liver cancer patients were different from patients suffering hepatic encephalopathy, cirrhosis, and hepatitis with significantly increased serum SA levels (Table 1). Among the 64 diseases, increased serum SA levels were the best biomarker for patients with pancreatitis, esophagus cancer, lung cancer, intracranial hemorrhage, uremia, and ovarian cancer with their Log10 p values >289 (Table 1). Based on overall data shown in Table 1 and Figs. 1 and 2, we could conclude that increased serum SA levels were not only associated with cancers but also associated with many different types of diseases. In addition, decreased serum SA levels were associated with liver diseases (Table 1), which suggested that liver might be the main production site for SA-containing glycoproteins in the blood circulation.59 Chronic hepatitis is one of the causes of cirrhosis and liver cancer. HBV infection ! chronic hepatitis !cirrhosis!hepatocarcinoma is the most important pathogenesis of liver cancer in China. Chronic hepatitis included chronic hepatitis B, chronic hepatitis C, autoimmune hepatitis, alcoholic liver disease, drug-induced hepatitis, and other types. Gruszewska et al.45 have reported that the serum SA levels in patients with chronic hepatitis B, chronic hepatitis C, and chronic nonviral hepatitis are decreased compared to that in healthy controls. In our study, serum SA levels in patients with unclassified hepatitis were lower than that in healthy controls, which were consistent with the published report.45 Cirrhosis is a high risk factor for liver cancer. When the liver is still able to carry out most or all of its functions, it is said that the liver is able to compensate for the damage. Cirrhosis at this stage is called compensated cirrhosis. Once the liver can no longer carry out its function, it is classified
The serum SA levels are significantly increased in sepsis
345
as decompensated cirrhosis. Matsuzaki et al.60 found that the serum SA levels in patients with compensated cirrhosis are significantly lower than that in healthy controls and are further reduced in patients with decompensated cirrhosis. Stefenelli et al. reported that the serum SA levels in patients with extensive cirrhosis continue to decline, especially in patients with serious complications.43 Furthermore, Arif et al.44 showed that the serum SA levels are not significantly different from that of healthy controls but the serum SA levels are significantly increased as the disease progresses. Their observation was explained by the increased production of the acute phase protein fibrinogen by the liver since fibrinogen is highly sialylated protein.45 All these results were consistent with our observation and supported the notion that liver might be the major production site for SA-containing glycoproteins in the blood circulation. Remarkably, the serum SA levels were significantly increased in liver cancer patients (Table 1), which suggested that liver cancer cells might compensate for the lost function of liver due to the decompensated cirrhosis and start to spill SA-containing glycoproteins into the blood circulation to start a new phase of the liver disease. Indeed, patients with sepsis had the similar characteristics. Sepsis is a life-threatening condition that arises when the body’s response to infection causes injury to its own tissues and organs.61 The risk of death from sepsis is 30%, from severe sepsis is 50%, and from septic shock is 80%.62 Our data showed that patients with sepsis had the highest mean and median serum SA levels and the highest SD value with moderate Log10 p value of 67.41 among the 64 diseases (Table 1) due to the extremely high and low serum SA levels in sepsis patients. We hypothesized that such fluctuation in the serum SA levels might reflect the compensated and decompensated period of this life threatening disease. We are currently testing our hypothesis and hope such information will help personalized diagnosis and care for patients with this devastating disease.
Acknowledgment This research was supported by the Natural Science Foundation of China (Grant 81672585), Key Technology Fund of Shandong Province (Grant 2016ZDJS07A07), the Taishan Scholar Fellowship, and the “Double First Class fund” of Shandong Province in China to L.Z.
Conflict of interest The authors declare no conflict of interest.
346
Xiaodan Huang et al.
References 1. Apweiler R, Hermjakob H, Sharon N. On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochim Biophys Acta. 1999;1473(1):4–8. 2. Zhang L. Glycosaminoglycan (GAG) biosynthesis and GAG-binding proteins. Prog Mol Biol Transl Sci. 2010;93:1–17. 3. Bi S, Baum LG. Sialic acids in T cell development and function. Biochim Biophys Acta. 2009;1790(12):1599–1610. 4. Varki A. Sialic acids in human health and disease. Trends Mol Med. 2008;14(8):351–360. 5. Collins BE, Blixt O, DeSieno AR, Bovin N, Marth JD, Paulson JC. Masking of CD22 by cis ligands does not prevent redistribution of CD22 to sites of cell contact. Proc Natl Acad Sci USA. 2004;101(16):6104–6109. 6. Varki A. Biological roles of oligosaccharides: all of the theories are correct. Glycobiology. 1993;3(2):97–130. 7. Varki A. Biological roles of glycans. Glycobiology. 2017;27(1):3–49. 8. Schauer R. Sialic acids as regulators of molecular and cellular interactions. Curr Opin Struct Biol. 2009;19(5):507–514. 9. Yuki N. Carbohydrate mimicry: a new paradigm of autoimmune diseases. Curr Opin Immunol. 2005;17(6):577–582. 10. Hughes RAC, Cornblath DR. Guillain-barre syndrome. Lancet. 2005;366(9497): 1653–1666. 11. Zhou J, Wang D, Gao R, et al. Biological features of novel avian influenza A (H7N9) virus. Nature. 2013;499(7459):500–503. 12. Hai W, Goda T, Takeuchi H, et al. Specific recognition of human influenza virus with PEDOT bearing sialic acid-terminated trisaccharides. ACS Appl Mater Interfaces. 2017;9(16):14162–14170. 13. Varki A, Gagneux P. Multifarious roles of sialic acids in immunity. Ann N Y Acad Sci. 2012;1253:16–36. 14. Malykh YN, Schauer R, Shaw L. N-glycolylneuraminic acid in human tumours. Biochimie. 2001;83(7):623–634. 15. Gruber S, Hendrikx T, Tsiantoulas D, et al. Sialic acid-binding immunoglobulin-like lectin G promotes atherosclerosis and liver inflammation by suppressing the protective functions of B-1 cells. Cell Rep. 2016;14(10):2348–2361. 16. El Maarouf A, Petridis A, Rutishauser U. Use of polysialic acid in repair of the central nervous system. Proc Natl Acad Sci USA. 2006;103(45):16989–16994. 17. Gascon E, Vutskits L, Kiss JZ. Polysialic acid-neural cell adhesion molecule in brain plasticity: from synapses to integration of new neurons. Brain Res Rev. 2007;56(1): 101–118. 18. Hildebrandt H, M€ uhlenhoff M, Gerardy-Schahn R. Polysialylation of NCAM. Adv Exp Med Biol. 2010;663:95–109. 19. Tettamanti G, Bonali F, Marchesini S, Zambotti V. A new procedure for the extraction, purification and fractionation of brain gangliosides. Biochim Biophys Acta. 1973;296(1): 160–170. 20. Schnaar RL. Brain gangliosides in axon-myelin stability and axon regeneration. FEBS Lett. 2010;584(9):1741–1747. 21. Colsch B, Jackson SN, Dutta S, Woods AS. Molecular microscopy of brain gangliosides: illustrating their distribution in hippocampal cell layers. ACS Chem Neurosci. 2011;2(4): 213–222. 22. Seki T, Arai Y. Temporal and spacial relationships between PSA-NCAM-expressing, newly generated granule cells, and radial glia-like cells in the adult dentate gyrus. J Comp Neurol. 1999;410(3):503–513.
The serum SA levels are significantly increased in sepsis
347
23. Seki T. Hippocampal adult neurogenesis occurs in a microenvironment provided by PSA-NCAM-expressing immature neurons. J Neurosci Res. 2002;69(6):772–783. 24. Singh R, Arya R. GNE myopathy and cell apoptosis A comparative mutation analysis. Mol Neurobiol. 2016;53(5):3088–3101. 25. Nishino I, Carrillo-Carrasco N, Argov Z. GNE myopathy: current update and future therapy. J Neurol Neurosurg Psychiatry. 2015;86(4):385–392. 26. Verheijen FW, Verbeek E, Aula N, et al. A new gene, encoding an anion transporter, is mutated in sialic acid storage diseases. Nat Genet. 1999;23(4):462–465. 27. Kleta R, Aughton DJ, Rivkin MJ, et al. Biochemical and molecular analyses of infantile free sialic acid storage disease in North American children. Am J Med Genet A. 2003;120A(1):28–33. 28. Wongkham S, Boonla C, Kongkham S, Wongkham C, Bhudhisawasdi V, Sripa B. Serum total sialic acid in cholangiocarcinoma patients: an ROC curve analysis. Clin Biochem. 2001;34(7):537–541. 29. Patel PS, Rawal GN, Balar DB. Importance of serum sialic acid and lactate dehydrogenase in diagnosis and treatment monitoring of cervical cancer patients. Gynecol Oncol. 1993;50(3):294–299. 30. Sebzda T, Saleh Y, Gburek J, et al. Total and lipid-bound plasma sialic acid as diagnostic markers in colorectal cancer patients correlation with cathepsin B expression in progression to Dukes stage. J Exp Ther Oncol. 2006;5(3):223–229. 31. Hogan-Ryan A, Fennelly JJ, Jones M, Cantwell B, Duffy MJ. Serum sialic acid and CEA concentrations in human breast cancer. Br J Cancer. 1980;41(4):587–592. 32. Vajaria BN, Patel KR, Begum R, et al. Evaluation of serum and salivary total sialic acid and alpha-l-fucosidase in patients with oral precancerous conditions and oral cancer. Oral Surg Oral Med Oral Pathol Oral Radiol. 2013;115(6):764–771. 33. Zhang Z, Wuhrer M, Holst S. Serum sialylation changes in cancer. Glycoconj J. 2018;35(2):139–160. 34. Almaraz RT, Tian Y, Bhattarcharya R, et al. Metabolic flux increases glycoprotein sialylation implications for cell adhesion and cancer metastasis. Mol Cell Proteomics. 2012;11(7):M112.017558. 35. Blain PG, Mucklow JC, Rawlins MD, Roberts DF, Routledge PA, Shand DG. Determinants of plasma alpha 1-acid glycoprotein (AAG) concentrations in health. Br J Clin Pharmacol. 1985;20(5):500–502. 36. Dage JL, Ackermann BL, Halsall HB. Site localization of sialyl Lewis(x) antigen on alpha1-acid glycoprotein by high performance liquid chromatography-electrospray mass spectrometry. Glycobiology. 1998;8(8):755–760. 37. Millar JS. The sialylation of plasma lipoproteins. Atherosclerosis. 2001;154(1):1–13. 38. Tertov VV, Kaplun VV, Sobenin IA, Boytsova EY, Bovin NV, Orekhov AN. Human plasma trans-sialidase causes atherogenic modification of low density lipoprotein. Atherosclerosis. 2001;159(1):103–115. 39. Przybysz M, Maszczak D, Borysewicz K, Szechinski J, Katnik-Prastowska I. Relative sialylation and fucosylation of synovial and plasma fibronectins in relation to the progression and activity of rheumatoid arthritis. Glycoconj J. 2007;24(9):543–550. 40. Alvi MH, Amer NA, Sumerin I. Serum 5-nucleotidase and serum sialic acid in pregnancy. Obstet Gynecol. 1988;72(2):171–174. 41. Goni M, Sayeed M, Shah GM, Hussain T. Serum sialic acid levels in normal pregnant and non-pregnant women. Indian J Physiol Pharmacol. 1981;25(4):356–360. 42. Jansen BC, Bondt A, Reiding KR, et al. Pregnancy-associated serum N-glycome changes studied by high-throughput MALDI-TOF-MS. Sci Rep. 2016;6:23296. 43. Stefenelli N, Klotz H, Engel A, Bauer P. Serum sialic acid in malignant tumors, bacterial infections, and chronic liver diseases. J Cancer Res Clin Oncol. 1985;109(1):55–59.
348
Xiaodan Huang et al.
44. Arif S, Najeeb-ul-Haq, Hanif R, Khan AS, Jamil-ur-Rehman, Mufti TA. Variations of serum sialic acid level in liver cirrhosis. J Ayub Med Coll Abbottabad. 2005;17(3):54–57. 45. Gruszewska E, Cylwik B, Panasiuk A, Szmitkowski M, Flisiak R, Chrostek L. Total and free serum sialic acid concentration in liver diseases. Biomed Res Int. 2014;2014:876096. 46. Winzler RJ. Determination of serum glycoproteins. Methods Biochem Anal. 1955;2: 279–311. 47. Macbeth RA, Bekesi JG. Oxygen consumption and anaerobic glycolysis of human malignant and normal tissue. Cancer Res. 1962;22:244–248. 48. Brozmanova E, Skrovina B. Value of determination of sialic acid in bone tumors. Acta Chir Orthop Traumatol Cech. 1969;36(5):285–291. 49. Narayanan S. Sialic acid as a tumor marker. Ann Clin Lab Sci. 1994;24(4):376–384. 50. Teshima S, Tamai K, Hayashi Y, Emi S. New enzymatic determination of sialic acid in serum. Clin Chem. 1988;34(11):2291–2294. 51. Horiuchi T, Kurokawa T. New enzymatic endpoint assay of serum sialic acid. Clin Chim Acta. 1989;182(1):117–121. 52. Rohrer JS. Analyzing sialic acids using high-performance anion-exchange chromatography with pulsed amperometric detection. Anal Biochem. 2000;283(1):3–9. 53. Shukla AK, Scholz N, Reimerdes EH, Schauer R. High-performance liquid chromatography of N,O-acylated sialic acids. Anal Biochem. 1982;123(1):78–82. 54. Vliegenthart JFG, Kamerling JP. Gas-liquid chromatography and mass spectrometry of sialic acids. In: Sialic Acids. Springer; 1982:95–125. 55. Morimoto N, Nakano M, Kinoshita M, et al. Specific distribution of sialic acids in animal tissues as examined by LC-ESI-MS after derivatization with 1,2-diamino-4,5methylenedioxybenzene. Anal Chem. 2001;73(22):5422–5428. 56. Klein A, Diaz S, Ferreira I, Lamblin G, Roussel P, Manzi AE. New sialic acids from biological sources identified by a comprehensive and sensitive approach liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) of SIA quinoxalinones. Glycobiology. 1997;7(3):421–432. 57. Spichtig V, Rohfritsch P, Austin S. Lactose does not interfere with the analysis of sialic acids as their 1,2-diamino-4,5-methylenedioxybenzene derivatives. Anal Bioanal Chem. 2011;399(5):1917–1922. 58. Hara S, Takemori Y, Yamaguchi M, Nakamura M, Ohkura Y. Fluorometric highperformance liquid chromatography of N-acetyl- and N-glycolylneuraminic acids and its application to their microdetermination in human and animal sera, glycoproteins, and glycolipids. Anal Biochem. 1987;164(1):138–145. 59. Cylwik B, Chrostek L, Panasiuk A, Szmitkowski M. Serum total and free sialic acid in patients with chronic liver disease. Clin Chem Lab Med. 2010;48(1):137–139. 60. Matsuzaki S, Itakura M, Iwamura K, Kamiguchi H. Serum sialic acid levels in liver cirrhosis and liver cancer. Jap J Gastroenterol. 1981;78(12):2395–2401. 61. Singer M, Deutschman CS, Seymour CW, et al. The third international consensus definitions for sepsis and septic shock (sepsis-3). JAMA. 2016;315(8):801–810. 62. Jawad I, Luksic I, Rafnsson SB. Assessing available information on the burden of sepsis: global estimates of incidence, prevalence and mortality. J Glob Health. 2012;2(1):010404.
The serum SA levels are significantly increased in sepsis but decreased in cirrhosis Xiaodan Huanga,b, Qin Yaoc, Lijuan Zhanga,*, Zibin Tianb,*
a Systems Biology and Medicine Center for Complex Diseases, Affiliated Hospital of Qingdao University, Qingdao, China b Department of Internal Medicine, Affiliated Hospital of Qingdao University, Qingdao, China c Department of Gynecology, Affiliated Hospital of Qingdao University, Qingdao, China *Corresponding authors: e-mail address: [email protected]; [email protected]
Contents 1. Introduction 2. Serum/plasma SA concentration measurement 3. Serum SA levels in 64 different types of diseases 3.1 Methods 3.2 Results 4. Discussions and conclusions Acknowledgment Conflict of interest References
336 338 339 339 339 343 345 345 346
Abstract Most of proteins in human blood circulation are glycoproteins with one or more covalently linked N- or O-linked glycans. Sialic acid (SA) generally occurs as the terminal monosaccharide on the glycans. SA in glycoproteins modulates a wide range of physiological and pathological processes and has been routinely measured in hospital since 1950s. Increased serum SA levels have been associated with different types of cancers. However, a systematic comparison of the serum SA levels in different types of human diseases has not been reported. In current study, 160,537 clinical lab test results of serum SA levels from healthy individuals and patients with 64 different types of diseases during the past 5 years in our hospital were retrieved and analyzed. Based on the mean (SD), median, and p (Log10 p) values, we found that patients suffering 55 different types of cancer and noncancer diseases such as sepsis, pancreatitis, bone cancer, rheumatoid arthritis, pancreatic cancer, and encephalitis had significantly (p < 0.05, Log10 p > 1.30) increased median serum SA levels whereas patients suffering hepatic encephalopathy, cirrhosis, renal cyst, and hepatitis had significantly decreased median serum SA levels compared to that of healthy controls. Moreover, the greatest increase in the mean (SD) and Log10 p values was observed in sepsis and pancreatitis, respectively,
Progress in Molecular Biology and Translational Science, Volume 162 ISSN 1877-1173 https://doi.org/10.1016/bs.pmbts.2019.01.009
#
2019 Elsevier Inc. All rights reserved.
335
336
Xiaodan Huang et al.
but not in cancers. Thus, the regulations of serum SA levels were much more complicated than previously assumed. Understanding the molecular mechanisms behind these observations would make serum SA a useful biomarker to facilitate personalized diagnosis and treatment for patients with different diseases.
1. Introduction More than 70% of human proteins are glycoproteins,1,2 which makes glycosylation the most common protein posttranslational modifications. N-linked and O-linked glycans are two most common types of glycans and are ubiquitous in all biological systems. Sialic acids (SAs) are a diverse family of naturally occurring 2-keto-3-deoxy-nonoinc acids with over 50 members. The most abundant SA in human is N-acetylneuraminic acid (Neu5Ac), which is one of the 10 monosaccharides found in N- or O-linked glycan chains. SA biosynthesis begins with conversion from UDP-N-acetylglucosamine (UDP-GlcNAc) by UDP-GlcNAc 2-epimerase to N-acetylmannosamine (ManNAc). CMP-Neu5Ac produced from ManNAc is the high energy donor for sialyltransferases that install SA to the terminal of the N- or O-linked glycan chains. In humans, 20 sialyltransferases transfer SA to other monosaccharides such as galactose (Ga1), N-acetylgalactosamine (N-GalNAc), GlcNAc, or other SA on the glycan chains with α-2,3, α-2,6, α-2,8, or α-2,9 linkage.3 Since no other monosaccharides can add to SA except that SA can add to itself, SA is the outmost monosaccharide of both N-linked and O-linked glycan chains.4 Being particularly prominent on cell surface and secreted molecules, for example, up to 10 million SAs per human erythrocyte and 100 mM estimated SA local concentrations on cell surface,5 SA-containing glycan chains are important players in many physiological and pathological processes.6,7 The negatively charged SA can mask the glycan chains on cell surface proteins by preventing the attack of glycosidase to maintain the integrity of epithelial cells from harmful substances and pathogens.8 At the same time, SA can help pathogenic microorganisms to escape the immune recognition of host, which happens when the pathogenic microorganisms are covered with SA-containing glycans. Yuki et al.9 and Hughes et al.10 reported such incidents when infected with Campylobacter jejuni, a bacterium containing SA-containing lipo-oligosaccharides on its wall, the patients suffered Guillian– Barre syndrome, an acute systemic peripheral neuropathy of quadriplegia.
The serum SA levels are significantly increased in sepsis
337
SA also plays an important role in several human viral infections since several viruses such as influenza viruses, adenoviruses, and rotaviruses can bind to host SA-containing glycans. More specifically, the influenza viruses have hemagglutinin activity glycoproteins (HA) on their surfaces that bind to specific SA-containing glycans on the surface of human erythrocytes and on the epithelial cell membranes of the upper respiratory tract.11,12 Such binding forms the basis of hemagglutination when the virus is mixed with erythrocytes. Such binding is also required for the entry of the influenza virus into epithelial cells of the upper respiratory tract. Widely used antiinfluenza drugs (oseltamivir and zanamivir) are SA analogs that interfere with release of newly generated viruses from infected cells by inhibiting the influenza viral enzyme neuraminidase.13 In human, the ligands for SA are diverse, such as hormones, lectins, antibodies, and inorganic ions, which mainly regulate the adhesion process of cells and cells in inflammation and immune response.14,15 The high density of SAs in extracellular membrane is important in maintaining normal filtration of the glomerular basement membrane.4 The brain tissue in the human contains more SAs than any other tissues. Poly-sialic acid (PSA) is uniquely present in brain tissues. PSA is a homogeneous polymer consisting of α-2, 8 linkages of SA and attach to the nervous system neuroadhesive molecules mainly through typical N-linked glycans. PSA can stimulate the growth of neuronal axons16,17 and assist nerve cell adhesion molecules (NCAM) complete synaptic effects for synaptic remodeling and stability.18 Gangliosides are a group of glycosphingolipids containing SA19 and composed of ceramide and SA-containing glycan chains. Gangliosides are concentrated in axonal terminals and synaptic terminals to maintain long-term stability of axon-myelin and to inhibit excessive axon regeneration after axonal injury.20 Other studies have found that the presence of large amounts of PSA-NCAM and neuroglycolipids in the hippocampus indicates that SA-containing glycans are closely related to the cognitive function of the hippocampus.21–23 About 60 genes are known to be involved in SA biology, where the GNE is responsible for SA synthesis. Mutations in GNE gene cause an autosomal recessive distal myopathy called conjugated vacuole distal myopathy (DMRV). In these patients, reduced ability to generate SAs leads to the damage of cell network.24 Under the electron microscope, rimmed vacuoles in the muscle fibers, abnormal protein deposition in the cells, and different diameters of the muscle fibers are observed in these patients.25 The clinical feature of DMRV is distal muscle atrophy and weakness. In addition, the mutation of SC17A5 gene can lead to SA storage diseases that are involved
338
Xiaodan Huang et al.
in the defect of transporting SA from lysosomes to cytosol.26,27 These patients usually have severe developmental delay, rough face, hepatosplenomegaly, and increased urinary free SA.27 Increased serum SA levels have been reported in patients with different types of cancers, such as, cholangiocarcinoma,28 cervical cancer,29 colorectal cancer,30 breast cancer,31 oral cancer,32 and other types of cancers.33 The increased serum SA levels are associated with the overexpression or increased activity of sialyltransferase in cancer cells.33 Almaraz et al. reported that by increasing the cellular levels of CMP-Neu5Ac, sialylation is greatly enhanced not in all but a subset of the glycans on specific proteins.34 Tumor cells that shed or secrete SA-containing glycoproteins into blood circulation might be another cause of elevated serum SA levels. However, increased serum SA levels are also reported in noncancer diseases, such as inflammation,35,36 atherosclerosis,37,38 rheumatoid arthritis,39 women during pregnancy,40–42 bacterial infections, and liver diseases.43–45 Since the serum SA levels in patients suffering different cancer or noncancer diseases have not been systematically studied and compared, such information would be needed to understand the molecular nature and the meaning of elevated serum SA levels. Thus, in current study a total of 160,537 clinical lab test results of serum SA levels from healthy individuals and patients with 64 different types of diseases during the past 5 years were retrieved and analyzed.
2. Serum/plasma SA concentration measurement Colorimetric assays are the detection methods for serum SA levels including resorcinol colorimetry and periodic acid/thiobarbiturate methods developed since 1950s.46–48 Between 98% and 99.5% of SA in serum is bound to the N- or O-linked glycans in glycoproteins and the rest is either free or bound to lipids in the form of gangliosides.49 Normal serum SA levels in healthy individuals are in the range of 1.6–2.8 mM while the free SA is in the range of 0.5–3 μM. Even though the colorimetric assays are not very sensitive, these assays are sufficient in measuring serum SA levels due to the mM ranges of SA in healthy individuals and increased levels in most of patients. With the rapid development of clinical laboratory techniques, many assays with increased accuracy and sensitivity have been developed. Enzymatic assay is one of the assays that can avoid the disadvantages of low sensitivity and many interfering factors compared with the colorimetric method. Teshima et al.50 and Hoviuchi et al.51 further improved the
The serum SA levels are significantly increased in sepsis
339
traditional enzymatic method to reduce the interference of pyruvic acid on SA measurement. Chromatographic methods can separate SA from other interfering compounds and quantitatively measure the serum SA levels. The high performance anion chromatography-pulse amperometric detection (HPAEC–PAD),52 high performance liquid chromatographic (HPLC),53 gas chromatography with mass spectrometry (GC–MS),54 and liquid chromatography coupled mass spectrum method (LC–MS)55,56 are some of the currently used chromatographic methods.55–58
3. Serum SA levels in 64 different types of diseases 3.1 Methods We have collected the lab data of serum SA levels of both healthy individuals and patients with clinically defined various diseases from the clinical laboratory of Affiliated Hospital of Qingdao University during the past 5 years. Each type of disease that has more than 30 independent test results for serum SA levels was included in current study. As a result, a total of 160,537 clinical lab results of SA from 64 different types of diseases were used in current study. Statistical analysis was performed by using SPSS version 19. Due to nonnormal distribution, Mann–Whitney test was used for statistical analysis. To compare the differences in p values among different types of diseases with that of healthy control, Log10 p values were calculated and used. Twosided test with a 5% significant level (p < 0.05 or Log10 p > 1.30) was considered as statistically significant.
3.2 Results The p value is used in the context of null hypothesis testing in order to quantify the idea of statistical significance of evidence. In essence, the statistical significance is assumed valid when the p values are far apart from that of control values. Based on the data collected, the mean (SD), median, and Log10 p values of serum SA levels for each type of diseases were listed in Table 1. The mean serum SA value is 58.00 U/mL in healthy controls. The data in Table 1 showed that 55/64 diseases in addition to healthy elderlies (>65 years old) had their median values significantly higher than that of healthy controls. In contrast, patients with hepatic encephalopathy, cirrhosis, renal cyst, hepatitis, and bladder stone had their median values lower than that of healthy controls, but for patient with bladder stone, the decreased median values had no statistical significance (Log10 p values
340
Xiaodan Huang et al.
Table 1 The mean, median, and Log10 p values of serum SA levels (U/mL) for healthy controls and patients with cancer and noncancer diseases. 2Log10 SA # of tests Mean (SD) Median p value
Sepsis
278
78.56 (22.21)
79.00
67.41
Pancreatitis
1708
76.46 (15.54)
75.00
>289
Esophagus cancer
3599
74.65 (16.59)
71.70
>289
Bone cancer
108
71.25 (15.30)
71.00
20.15
Rheumatoid arthritis
256
73.00 (14.43)
70.15
71.30
Pancreatic cancer
910
71.41 (13.92)
70.00
168.67
Encephalitis
640
70.22 (14.27)
69.00
98.00
Lung fibrosis
318
70.30 (13.35)
68.35
63.01
Ankylosing spondylitis
69
69.13 (11.09)
68.30
16.24
Brain trauma
539
71.44 (16.95)
68.00
71.19
Lung cancer
8840
71.36 (14.70)
68.00
>289
Intracranial hemorrhage
3345
70.63 (14.33)
68.00
>289
Aplastic anemia
671
70.26 (14.75)
67.50
100.35
Lupus erythematosus
835
68.07 (10.52)
67.50
138.24
Uremia
3785
69.39 (11.65)
67.30
>289
Gout
1366
69.41 (14.34)
67.00
162.52
Ovarian cancer
2019
70.00 (12.93)
66.90
>289
Chronic obstructive PD
1534
67.87 (12.36)
66.00
169.57
Acute myocardial infarction
2490
67.41 (12.52)
65.20
205.04
Azotemia
382
66.88 (11.04)
65.15
50.75
Colon cancer
5534
67.56 (13.56)
65.00
288.86
Diabetic nephropathy
505
66.52 (10.74)
65.00
62.45
Cystitis
49
65.12 (8.05)
65.00
8.03
Preeclampsia
963
65.69 (7.87)
64.80
140.76
Anemia
1739
67.55 (15.17)
64.60
107.60
Nephrotic syndrome
2660
65.34 (9.37)
64.40
216.59
Gastric cancer
11,755
67.66 (15.29)
64.30
286.43
341
The serum SA levels are significantly increased in sepsis
Table 1 The mean, median, and Log10 p values of serum SA levels (U/mL) for healthy controls and patients with cancer and noncancer diseases.—cont’d 2Log10 SA # of tests Mean (SD) Median p value
Endometrial cancer
1035
66.04 (9.77)
64.10
126.63
Gastric ulcer
289
66.78 (14.85)
64.00
17.60
Liver cancer
237
66.03 (13.66)
64.00
14.66
Postbreast surgery
32
67.56 (11.07)
63.95
6.51
Necrosis of femoral head
166
66.26 (11.13)
63.85
20.35
Cervical cancer
1854
65.34 (9.71)
63.80
163.09
Bone fracture
1938
65.44 (11.82)
63.30
111.97
Myeloproliferative disorder
1113
67.03 (16.08)
63.10
48.45
Multiple myeloma
1938
67.08 (18.05)
63.00
66.06
Lymphoma
4305
66.33 (13.52)
63.00
187.33
Rectum cancer
7268
65.82 (13.33)
63.00
194.30
Alzheimer’s disease
95
65.52 (11.61)
63.00
10.32
Nephritis
1532
64.81 (10.87)
63.00
93.77
Psoriasis
98
63.47 (10.09)
62.25
6.32
Asthma
600
63.99 (10.76)
62.00
31.46
Kidney cancer
1282
63.84 (10.48)
61.70
58.75
Acute cerebral infarction
9286
63.50 (10.71)
61.70
151.37
Knee-joint degenerative diseases
432
62.20 (7.83)
61.50
19.35
Bladder cancer
941
63.18 (12.31)
61.00
19.40
Osteoarthritis
74
63.71 (10.02)
60.75
5.00
Breast cancer
3737
61.42 (6.64)
60.70
82.46
Hyperuricemia
118
61.21 (7.24)
60.55
4.04
Leukemia
7627
63.64 (14.29)
60.50
48.74
Coronary heart disease
20,613
62.30 (10.44)
60.50
99.34
Type 2 diabetes mellitus
10,022
61.01 (9.65)
59.40
30.61
Breast lumps
81
60.07 (6.54)
59.30
1.54
Cerebrovascular disease
4560
60.72 (9.54)
59.10
15.27 Continued
342
Xiaodan Huang et al.
Table 1 The mean, median, and Log10 p values of serum SA levels (U/mL) for healthy controls and patients with cancer and noncancer diseases.—cont’d 2Log10 SA # of tests Mean (SD) Median p value
Cerebral arteriosclerosis
751
59.91 (8.21)
59.00
2.64
Osteoporosis
247
59.63 (9.15)
59.00
0.68
Wilms’ tumor
225
61.03 (11.33)
58.50
0.92
Healthy controls >65 years old
531
59.23 (6.35)
58.30
1.74
Cerebral ischemia
2194
59.18 (8.10)
58.10
0.59
Gastritis
2633
58.99 (7.99)
58.00
0.65
Healthy controls
6053
58.61 (6.24)
58.00
0.00
Bladder stone
157
59.93 (10.13)
57.80
0.41
Hepatitis
2831
56.40 (9.54)
56.90
20.86
Renal cyst
496
56.93 (7.64)
56.25
9.32
Cirrhosis
6703
50.33 (12.03)
50.40
>289
Hepatic encephalopathy
77
47.83 (13.08)
45.50
13.44
>1.30). The following diseases had the highest mean values in a descending order: sepsis (78.56), pancreatitis (76.46), esophagus cancer (74.65), rheumatoid arthritis (73.00). The following diseases had the highest SD values: sepsis (22.21), multiple myeloma (18.05), brain trauma (16.95), esophagus cancer (16.59), and myeloproliferative disorder (16.08). To make the data more intuitive, scatter diagram of serum SA levels in the 48 diseases with Log10 p values >15 in comparison to the healthy controls was plotted with lower quartile (25%), median (50%), and upper quartile (75%) marked and showed in Fig. 1. Patients with sepsis had the highest median value followed by patients with pancreatitis, esophagus cancer, bone cancer, rheumatoid arthritis, and pancreatic cancer whereas patients suffering cirrhosis and hepatitis had the lowest median values. The Log10 p values were further plotted and showed in Fig. 2. The highest Log10 p values mean the highest difference in the patients as a group compared to the healthy controls as another group. According to the Log10 p values, the increased serum SA levels worked best as biomarkers for patients with pancreatitis, esophagus cancer, lung cancer, intracranial hemorrhage, uremia, and ovarian cancer among other diseases (Table 1 and Fig. 2).
The serum SA levels are significantly increased in sepsis
343
Fig. 1 Serum SA levels in 48 different types of diseases. The data were sorted in a descending order of the median values. Serum SA levels for each type of disease with lower quartile (25%), median (50%), and upper quartile (75%) ranges were marked in red.
Fig. 2 Log10 p values of SA in 48 different types of diseases compared to that of healthy controls. When the serum SA levels were lower than that of healthy controls, blue color was used in the plot whereas when the serum AFP levels were higher than that of healthy controls, red color was used in the plot.
4. Discussions and conclusions Aberrant glycosylation and sialylation of glycan chains has been recognized as one of the hallmarks of cancers and abnormal immune systems.4,6,7,13,49 Increased serum SA levels have been tested routinely in hospital mainly for the purposes of cancer monitoring and prognosis.33 However, based on data obtained from the serum SA levels from 154,484 patients with 64 clinically defined diseases as well as serum SA levels from 6053 healthy individuals (Table 1), our results showed that: (1) The median serum SA levels in patients with 55 different types of diseases were significantly increased (Log10 p > 1.30) compared to that in healthy controls
344
Xiaodan Huang et al.
(Table 1); (2) Patients with sepsis had the highest mean serum SA levels and the highest SD value among the 64 diseases (Table 1); (3) Based on the Log10 p values, the serum SA levels worked best as biomarkers for patients with pancreatitis, esophagus cancer, lung cancer, intracranial hemorrhage, uremia, ovarian cancer, colon cancer, gastric cancer, nephrotic syndrome, acute myocardial infarction, rectum cancer, and lymphoma among many other cancers and noncancer diseases (Table 1 and Fig. 2); (4) Patients with hepatic encephalopathy, cirrhosis, and hepatitis had both lowest mean and median serum SA levels. Among the three liver diseases, patients with hepatic encephalopathy had the lowest mean but the biggest SD values. Therefore, the increased serum SA levels were not specifically associated with cancers but the decreased serum SA levels were directly associated with liver diseases in the measures of the mean (SD), median, or Log10 p values of our current studies (Table 1 and Figs. 1 and 2); (5) Liver cancer patients were different from patients suffering hepatic encephalopathy, cirrhosis, and hepatitis with significantly increased serum SA levels (Table 1). Among the 64 diseases, increased serum SA levels were the best biomarker for patients with pancreatitis, esophagus cancer, lung cancer, intracranial hemorrhage, uremia, and ovarian cancer with their Log10 p values >289 (Table 1). Based on overall data shown in Table 1 and Figs. 1 and 2, we could conclude that increased serum SA levels were not only associated with cancers but also associated with many different types of diseases. In addition, decreased serum SA levels were associated with liver diseases (Table 1), which suggested that liver might be the main production site for SA-containing glycoproteins in the blood circulation.59 Chronic hepatitis is one of the causes of cirrhosis and liver cancer. HBV infection ! chronic hepatitis !cirrhosis!hepatocarcinoma is the most important pathogenesis of liver cancer in China. Chronic hepatitis included chronic hepatitis B, chronic hepatitis C, autoimmune hepatitis, alcoholic liver disease, drug-induced hepatitis, and other types. Gruszewska et al.45 have reported that the serum SA levels in patients with chronic hepatitis B, chronic hepatitis C, and chronic nonviral hepatitis are decreased compared to that in healthy controls. In our study, serum SA levels in patients with unclassified hepatitis were lower than that in healthy controls, which were consistent with the published report.45 Cirrhosis is a high risk factor for liver cancer. When the liver is still able to carry out most or all of its functions, it is said that the liver is able to compensate for the damage. Cirrhosis at this stage is called compensated cirrhosis. Once the liver can no longer carry out its function, it is classified
The serum SA levels are significantly increased in sepsis
345
as decompensated cirrhosis. Matsuzaki et al.60 found that the serum SA levels in patients with compensated cirrhosis are significantly lower than that in healthy controls and are further reduced in patients with decompensated cirrhosis. Stefenelli et al. reported that the serum SA levels in patients with extensive cirrhosis continue to decline, especially in patients with serious complications.43 Furthermore, Arif et al.44 showed that the serum SA levels are not significantly different from that of healthy controls but the serum SA levels are significantly increased as the disease progresses. Their observation was explained by the increased production of the acute phase protein fibrinogen by the liver since fibrinogen is highly sialylated protein.45 All these results were consistent with our observation and supported the notion that liver might be the major production site for SA-containing glycoproteins in the blood circulation. Remarkably, the serum SA levels were significantly increased in liver cancer patients (Table 1), which suggested that liver cancer cells might compensate for the lost function of liver due to the decompensated cirrhosis and start to spill SA-containing glycoproteins into the blood circulation to start a new phase of the liver disease. Indeed, patients with sepsis had the similar characteristics. Sepsis is a life-threatening condition that arises when the body’s response to infection causes injury to its own tissues and organs.61 The risk of death from sepsis is 30%, from severe sepsis is 50%, and from septic shock is 80%.62 Our data showed that patients with sepsis had the highest mean and median serum SA levels and the highest SD value with moderate Log10 p value of 67.41 among the 64 diseases (Table 1) due to the extremely high and low serum SA levels in sepsis patients. We hypothesized that such fluctuation in the serum SA levels might reflect the compensated and decompensated period of this life threatening disease. We are currently testing our hypothesis and hope such information will help personalized diagnosis and care for patients with this devastating disease.
Acknowledgment This research was supported by the Natural Science Foundation of China (Grant 81672585), Key Technology Fund of Shandong Province (Grant 2016ZDJS07A07), the Taishan Scholar Fellowship, and the “Double First Class fund” of Shandong Province in China to L.Z.
Conflict of interest The authors declare no conflict of interest.
346
Xiaodan Huang et al.
References 1. Apweiler R, Hermjakob H, Sharon N. On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochim Biophys Acta. 1999;1473(1):4–8. 2. Zhang L. Glycosaminoglycan (GAG) biosynthesis and GAG-binding proteins. Prog Mol Biol Transl Sci. 2010;93:1–17. 3. Bi S, Baum LG. Sialic acids in T cell development and function. Biochim Biophys Acta. 2009;1790(12):1599–1610. 4. Varki A. Sialic acids in human health and disease. Trends Mol Med. 2008;14(8):351–360. 5. Collins BE, Blixt O, DeSieno AR, Bovin N, Marth JD, Paulson JC. Masking of CD22 by cis ligands does not prevent redistribution of CD22 to sites of cell contact. Proc Natl Acad Sci USA. 2004;101(16):6104–6109. 6. Varki A. Biological roles of oligosaccharides: all of the theories are correct. Glycobiology. 1993;3(2):97–130. 7. Varki A. Biological roles of glycans. Glycobiology. 2017;27(1):3–49. 8. Schauer R. Sialic acids as regulators of molecular and cellular interactions. Curr Opin Struct Biol. 2009;19(5):507–514. 9. Yuki N. Carbohydrate mimicry: a new paradigm of autoimmune diseases. Curr Opin Immunol. 2005;17(6):577–582. 10. Hughes RAC, Cornblath DR. Guillain-barre syndrome. Lancet. 2005;366(9497): 1653–1666. 11. Zhou J, Wang D, Gao R, et al. Biological features of novel avian influenza A (H7N9) virus. Nature. 2013;499(7459):500–503. 12. Hai W, Goda T, Takeuchi H, et al. Specific recognition of human influenza virus with PEDOT bearing sialic acid-terminated trisaccharides. ACS Appl Mater Interfaces. 2017;9(16):14162–14170. 13. Varki A, Gagneux P. Multifarious roles of sialic acids in immunity. Ann N Y Acad Sci. 2012;1253:16–36. 14. Malykh YN, Schauer R, Shaw L. N-glycolylneuraminic acid in human tumours. Biochimie. 2001;83(7):623–634. 15. Gruber S, Hendrikx T, Tsiantoulas D, et al. Sialic acid-binding immunoglobulin-like lectin G promotes atherosclerosis and liver inflammation by suppressing the protective functions of B-1 cells. Cell Rep. 2016;14(10):2348–2361. 16. El Maarouf A, Petridis A, Rutishauser U. Use of polysialic acid in repair of the central nervous system. Proc Natl Acad Sci USA. 2006;103(45):16989–16994. 17. Gascon E, Vutskits L, Kiss JZ. Polysialic acid-neural cell adhesion molecule in brain plasticity: from synapses to integration of new neurons. Brain Res Rev. 2007;56(1): 101–118. 18. Hildebrandt H, M€ uhlenhoff M, Gerardy-Schahn R. Polysialylation of NCAM. Adv Exp Med Biol. 2010;663:95–109. 19. Tettamanti G, Bonali F, Marchesini S, Zambotti V. A new procedure for the extraction, purification and fractionation of brain gangliosides. Biochim Biophys Acta. 1973;296(1): 160–170. 20. Schnaar RL. Brain gangliosides in axon-myelin stability and axon regeneration. FEBS Lett. 2010;584(9):1741–1747. 21. Colsch B, Jackson SN, Dutta S, Woods AS. Molecular microscopy of brain gangliosides: illustrating their distribution in hippocampal cell layers. ACS Chem Neurosci. 2011;2(4): 213–222. 22. Seki T, Arai Y. Temporal and spacial relationships between PSA-NCAM-expressing, newly generated granule cells, and radial glia-like cells in the adult dentate gyrus. J Comp Neurol. 1999;410(3):503–513.
The serum SA levels are significantly increased in sepsis
347
23. Seki T. Hippocampal adult neurogenesis occurs in a microenvironment provided by PSA-NCAM-expressing immature neurons. J Neurosci Res. 2002;69(6):772–783. 24. Singh R, Arya R. GNE myopathy and cell apoptosis A comparative mutation analysis. Mol Neurobiol. 2016;53(5):3088–3101. 25. Nishino I, Carrillo-Carrasco N, Argov Z. GNE myopathy: current update and future therapy. J Neurol Neurosurg Psychiatry. 2015;86(4):385–392. 26. Verheijen FW, Verbeek E, Aula N, et al. A new gene, encoding an anion transporter, is mutated in sialic acid storage diseases. Nat Genet. 1999;23(4):462–465. 27. Kleta R, Aughton DJ, Rivkin MJ, et al. Biochemical and molecular analyses of infantile free sialic acid storage disease in North American children. Am J Med Genet A. 2003;120A(1):28–33. 28. Wongkham S, Boonla C, Kongkham S, Wongkham C, Bhudhisawasdi V, Sripa B. Serum total sialic acid in cholangiocarcinoma patients: an ROC curve analysis. Clin Biochem. 2001;34(7):537–541. 29. Patel PS, Rawal GN, Balar DB. Importance of serum sialic acid and lactate dehydrogenase in diagnosis and treatment monitoring of cervical cancer patients. Gynecol Oncol. 1993;50(3):294–299. 30. Sebzda T, Saleh Y, Gburek J, et al. Total and lipid-bound plasma sialic acid as diagnostic markers in colorectal cancer patients correlation with cathepsin B expression in progression to Dukes stage. J Exp Ther Oncol. 2006;5(3):223–229. 31. Hogan-Ryan A, Fennelly JJ, Jones M, Cantwell B, Duffy MJ. Serum sialic acid and CEA concentrations in human breast cancer. Br J Cancer. 1980;41(4):587–592. 32. Vajaria BN, Patel KR, Begum R, et al. Evaluation of serum and salivary total sialic acid and alpha-l-fucosidase in patients with oral precancerous conditions and oral cancer. Oral Surg Oral Med Oral Pathol Oral Radiol. 2013;115(6):764–771. 33. Zhang Z, Wuhrer M, Holst S. Serum sialylation changes in cancer. Glycoconj J. 2018;35(2):139–160. 34. Almaraz RT, Tian Y, Bhattarcharya R, et al. Metabolic flux increases glycoprotein sialylation implications for cell adhesion and cancer metastasis. Mol Cell Proteomics. 2012;11(7):M112.017558. 35. Blain PG, Mucklow JC, Rawlins MD, Roberts DF, Routledge PA, Shand DG. Determinants of plasma alpha 1-acid glycoprotein (AAG) concentrations in health. Br J Clin Pharmacol. 1985;20(5):500–502. 36. Dage JL, Ackermann BL, Halsall HB. Site localization of sialyl Lewis(x) antigen on alpha1-acid glycoprotein by high performance liquid chromatography-electrospray mass spectrometry. Glycobiology. 1998;8(8):755–760. 37. Millar JS. The sialylation of plasma lipoproteins. Atherosclerosis. 2001;154(1):1–13. 38. Tertov VV, Kaplun VV, Sobenin IA, Boytsova EY, Bovin NV, Orekhov AN. Human plasma trans-sialidase causes atherogenic modification of low density lipoprotein. Atherosclerosis. 2001;159(1):103–115. 39. Przybysz M, Maszczak D, Borysewicz K, Szechinski J, Katnik-Prastowska I. Relative sialylation and fucosylation of synovial and plasma fibronectins in relation to the progression and activity of rheumatoid arthritis. Glycoconj J. 2007;24(9):543–550. 40. Alvi MH, Amer NA, Sumerin I. Serum 5-nucleotidase and serum sialic acid in pregnancy. Obstet Gynecol. 1988;72(2):171–174. 41. Goni M, Sayeed M, Shah GM, Hussain T. Serum sialic acid levels in normal pregnant and non-pregnant women. Indian J Physiol Pharmacol. 1981;25(4):356–360. 42. Jansen BC, Bondt A, Reiding KR, et al. Pregnancy-associated serum N-glycome changes studied by high-throughput MALDI-TOF-MS. Sci Rep. 2016;6:23296. 43. Stefenelli N, Klotz H, Engel A, Bauer P. Serum sialic acid in malignant tumors, bacterial infections, and chronic liver diseases. J Cancer Res Clin Oncol. 1985;109(1):55–59.
348
Xiaodan Huang et al.
44. Arif S, Najeeb-ul-Haq, Hanif R, Khan AS, Jamil-ur-Rehman, Mufti TA. Variations of serum sialic acid level in liver cirrhosis. J Ayub Med Coll Abbottabad. 2005;17(3):54–57. 45. Gruszewska E, Cylwik B, Panasiuk A, Szmitkowski M, Flisiak R, Chrostek L. Total and free serum sialic acid concentration in liver diseases. Biomed Res Int. 2014;2014:876096. 46. Winzler RJ. Determination of serum glycoproteins. Methods Biochem Anal. 1955;2: 279–311. 47. Macbeth RA, Bekesi JG. Oxygen consumption and anaerobic glycolysis of human malignant and normal tissue. Cancer Res. 1962;22:244–248. 48. Brozmanova E, Skrovina B. Value of determination of sialic acid in bone tumors. Acta Chir Orthop Traumatol Cech. 1969;36(5):285–291. 49. Narayanan S. Sialic acid as a tumor marker. Ann Clin Lab Sci. 1994;24(4):376–384. 50. Teshima S, Tamai K, Hayashi Y, Emi S. New enzymatic determination of sialic acid in serum. Clin Chem. 1988;34(11):2291–2294. 51. Horiuchi T, Kurokawa T. New enzymatic endpoint assay of serum sialic acid. Clin Chim Acta. 1989;182(1):117–121. 52. Rohrer JS. Analyzing sialic acids using high-performance anion-exchange chromatography with pulsed amperometric detection. Anal Biochem. 2000;283(1):3–9. 53. Shukla AK, Scholz N, Reimerdes EH, Schauer R. High-performance liquid chromatography of N,O-acylated sialic acids. Anal Biochem. 1982;123(1):78–82. 54. Vliegenthart JFG, Kamerling JP. Gas-liquid chromatography and mass spectrometry of sialic acids. In: Sialic Acids. Springer; 1982:95–125. 55. Morimoto N, Nakano M, Kinoshita M, et al. Specific distribution of sialic acids in animal tissues as examined by LC-ESI-MS after derivatization with 1,2-diamino-4,5methylenedioxybenzene. Anal Chem. 2001;73(22):5422–5428. 56. Klein A, Diaz S, Ferreira I, Lamblin G, Roussel P, Manzi AE. New sialic acids from biological sources identified by a comprehensive and sensitive approach liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) of SIA quinoxalinones. Glycobiology. 1997;7(3):421–432. 57. Spichtig V, Rohfritsch P, Austin S. Lactose does not interfere with the analysis of sialic acids as their 1,2-diamino-4,5-methylenedioxybenzene derivatives. Anal Bioanal Chem. 2011;399(5):1917–1922. 58. Hara S, Takemori Y, Yamaguchi M, Nakamura M, Ohkura Y. Fluorometric highperformance liquid chromatography of N-acetyl- and N-glycolylneuraminic acids and its application to their microdetermination in human and animal sera, glycoproteins, and glycolipids. Anal Biochem. 1987;164(1):138–145. 59. Cylwik B, Chrostek L, Panasiuk A, Szmitkowski M. Serum total and free sialic acid in patients with chronic liver disease. Clin Chem Lab Med. 2010;48(1):137–139. 60. Matsuzaki S, Itakura M, Iwamura K, Kamiguchi H. Serum sialic acid levels in liver cirrhosis and liver cancer. Jap J Gastroenterol. 1981;78(12):2395–2401. 61. Singer M, Deutschman CS, Seymour CW, et al. The third international consensus definitions for sepsis and septic shock (sepsis-3). JAMA. 2016;315(8):801–810. 62. Jawad I, Luksic I, Rafnsson SB. Assessing available information on the burden of sepsis: global estimates of incidence, prevalence and mortality. J Glob Health. 2012;2(1):010404.