The majority of studies conclude that AFP is not a useful diagnostic test for the detection of HCC30–33, but AFP continues to be used as the common tumor marker 34. Current studies examining the test characteristics of AFP for diagnosing HCC in patients with one or more risk factors have substantial methodological limitations, making it difficult to distinguish between falsely elevated AFP levels and tumors in benign liver diseases35,36.
In our study, the AFP level was identified as a risk factor for HCC development in HCV-infected patients, where its level is between 6 and 20 ng/ml. The present study showed that the level of AFP was increased in the early stages of HCC, but not in all HCC cases. AFP lacks its sensitivity in a small portion of early-detected HCC patients with an elevation in AFP level37. It’s a non-invasive predictive tumor marker of the progression of HCC in HCV patients, but it is difficult to estimate the sensitivity and specificity for this test clearly38. We need more straightforward non-invasive serological biomarkers that can be combined with / or replace AFP to significantly improve HCC diagnosis. The main objective is to uncover new biomarkers because of the low AFP sensitivity and specificity in diagnosis and follow-up of HCC among HCV-infected patients with or without DM.
Our results showed that type II DM patients had a significant increase in both plasma glucose and insulin levels. It means that our DM subjects suffer insulin resistance, due to elevated insulin levels despite the increased glucose level.
Insulin resistance can unfortunately worsen the disease state of the patient when associated with metabolic disease 39. As well as, fasting blood glucose levels were higher in HCV-infected patients than healthy controls, an observation that was reported before 40,41. This hyperglycemia could increase oxidative stress in the cells, which may initiate cancer development42–44.
In our study, blood glucose level was decreased in HCC patients, this showed an accordance with some previously reported observations 45,46. They revealed that the HCC group showed higher levels of insulin. Previous studies proved that chronic HCV infection may damage the pancreatic beta cells, leading to the death of these cells through multiple mechanisms47–49.
As well, insulin resistance is commonly associated with chronic liver diseases 50,51. Our study showed an agreement with some previous data assuming that insulin levels were higher in diabetic HCV infected patients and diabetic HCC patients, than patients with type II DM 45,52. Early studies suggested that hyperinsulinemia can accelerate the progression of HCC rather than hyperglycemia53, and even the treatment with insulin constitutes a risk factor for HCC54,55.
It was reported that hyperglycemic conditions would lead to a reduction in the average lifespan of RBCs and consequently would reduce RBCs count 56. We pursued biochemical pathways of glucose conversion into sorbitol, a polyol pathway (PP), in addition to AR and SDH expression to check whether they are involved in the pathogenesis of HCC among DM patients infected with HCV. The same parameters, in addition to plasma insulin, were used to prove the role of RBCs in the early prediction of HCC progression. Here, we called the AR and SDH as polyol profile, that was studied in DM type II in both plasma and RBCs to follow the variations in PP during the disease development and to see whether this profile can help as a possible early index for progression to HCC in diabetic patients infected with hepatitis C virus. As glucose uptake by the liver is not dependent on insulin, the glycation reaction would be enhanced in the liver by hyperglycemia 13,57.
AR is the rate-limiting enzyme in the polyol pathway, converting glucose to sorbitol using NADPH as a co-factor11. The second enzyme of the PP is SDH, catalyzing the conversion of sorbitol to fructose using NAD+ as a co-factor 58.
Our result showed that AR expression was significantly increased in DM59, Glucose to sorbitol conversion leads to NADPH cellular depletion which can induce oxidative stress by impairing glutathione metabolism60,61 increasing the cell death, and accumulation of intracellular sorbitol62. It was reported that AR activity was found to be significantly higher among HCV-infected and HCC patients63,64.
The hyperglycemic condition results in activation of the PP and increases the activity of SDH, leading to the formation of a large amount of fructose65, and the increase of NADH: NAD+ ratio 11,66. In our study, SDH content was increased with increasing glucose levels in both plasma and RBCs.
Increasing NADH/NAD+ ratio under hyperglycemic conditions, and excess NADH can be used as the substrate for NADH oxidase, which can lead to oxidative stress67,68.
SDH converts glucose to fructose in the pp and excessive metabolites as fructose-3-phosphate and 3-deoxyglucosone are produced, being more effective non-enzymatic glycation agents than glucose69. The flux of glucose through the PP would increase the production of AGEs, causing oxidative stress70–72. Also, fructose is a stronger glycating sugar than glucose 73–75. In our study SDH activity decreases in the diabetic group along with increased plasma and RBCs’ glucose levels.
In diabetes, fructose is overproduced in the body, as the PP consumes approximately 30% of blood glucose11. Overproduction of fructose leads to metabolic consequences. Thus, excess fructose can glycate proteins74, causing protein dysfunction. In the current work, plasma glucose level was decreased in the HCC group, and RBCs as well. High blood glucose level encourages sorbitol accumulation, preventing its conversion to fructose 58,76.
Our results showed that SDH activity was increased in HCV-infected patients, which is following with another registered study77. In our results, SDH contents were higher in HCV-infected patients than HCC patients, our results are in accordance with a previous study 78, suggesting that activity of SDH promotes migration and invasion of HCC-cells. A previous study suggested a critical tumor-suppressive role for SDH in HCC.
RBCs’ AR content was significantly higher in all groups than in plasma even in the control group. It was postulated that the contents of erythrocytic AR, as well as sorbitol may have a value as a quantitative trait to be included with other markers to establish a risk profile for the development of late diabetic complications79. There was a linear correlation between serum AFP levels and both AR and SDH contents in plasma and RBCs of studied groups. It was noticed that serum SDH levels of more than15 ng/ml was associated with shorter recurrence-free survival after surgical interventions to patients with HCC. Moreover, baseline serum SDH and alpha-fetoprotein (AFP) levels might better predict the recurrence of HCC, so, incorporating serum SDH along with AFP levels in clinical practice may elevate the predictability of prognosis in HCC patients80.
As well as, our work displayed a negative correlation between elevated plasma glucose with plasma SDH levels. The same was noticed in RBCs’ glucose and SDH values in the studied groups. Here we can translate these correlations in a way that RBCs can offer the same correlation instead of plasma, but a given benefit arises by using the short life-spanned vehicle (RBCs) which is greatly expressive for early changes than plasma. We previously assumed this benefit16. In all cases, defective SDH expression might adversely affect the prognosis towards late diabetic complications.
Conversely, a positive correlation between elevated plasma glucose with plasma AR level was observed. The same was noticed in RBCs’ glucose and AR values in studied groups. It was reported that high glucose levels elevated AR protein expression; although, this was not accompanied by apparent enzyme activation81. Correlations in our study were calculated by plotting each parameter against the other parameter as a stream in all groups to guess the association between these parameters as an absolute correlation in the studied groups.
Interestingly, our study revealed that a strong positive correlation is found between plasma and RBCs glucose in the studied groups. Recent literature agree that RBCs of DM patients are greatly affected by hyperglycemia in a manner that a plethora of adverse changes occurs in the RBCs including a correlative increased glucose content to hyperglycemia and late diabetic complications82.
Cancer activates the conversion of sorbitol to fructose promoting SDH expression. Previous studies also revealed that SDH expression was significantly elevated in HCV and HCC patients and elevations were prominent in RBCs than plasma, adding that SDH activity is significantly reduced in diabetic patients69.
These results to be more popularized, repetition on large scale multicenter is necessitated. Further studies including single nucleotide polymorphisms (SNPs) and RNA expression of both AR and SDH in both type I and type II DM patients to predict the future of late diabetic complications of these patients especially patients co-mordidified by hepatitis C or B infections.