Plasma Insulin/Erythrocytic Aldose Reductase Ratio as a Predictor for Hepatocellular Carcinoma Among Type II Diabetics and Hepatitis C Virus-Infected Patients

doi:10.21203/rs.3.rs-1220253/v1

Purpose

Hepatocellular carcinoma (HCC) is a possible oncogenic progression during persistent hepatitis C-infection +/- type II diabetes mellitus (DM). To investigate the plasma insulin, erythrocytic aldose reductase (AR) and sorbitol dehydrogenase (SDH) as possible predictive tools for HCC in hepatitis C-infected patients (HCV) +/- DM. Erythrocytes (RBCs) were adopted as a possible vehicle for pre-malignant variations being of short life span.

Methods

The study included 20 healthy control and 100 patients of 48–64 years old, divided into 5 equal groups as; type II DM, HCC, HCC with DM, DM- HCV infected and non-DM HCV infected. Plasma levels of AFP and insulin were measured.

Results

It showed an elevated AR, significant reduction of SDH in RBCs and plasma of DM patients. These values were greatly elevated among HCV, HCC, diabetic HCV, and diabetic HCC patients. All DM patients showed elevated insulin levels than normoglycemic controls.

Conclusion

The study substantiated the use of RBCs as a vehicle for early diagnostic markers better than plasma. We recommend the use of insulin/ erythrocytic AR ratio as a new laboratory marker for predicting HCC among type II diabetics or non-treated HCV-infected patients with control insulin/ erythrocytic AR ratio by each laboratory.

Plasma insulin/erythrocytic aldose reductase ratio

Sorbitol dehydrogenase

Type II Diabetes mellitus

Hepatocellular carcinoma

Hepatitis C virus.

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide¹, and the second cause leading to human cancer-related deaths ². It has a poor prognosis due to its rapid infiltrating power and complicating liver cirrhosis ³. Etiological factors of the disease include infection with the hepatitis B or C virus or exposure to high levels of dietary aflatoxin B1¹. Approximately, 75-80% of primary liver cancers are attributable to persistent viral infections. Cirrhosis is the major risk factor for HCC and cirrhotic patients should be screened for early detection ^{4 5}.

Although ultra-sonography (US) and conventional tumor markers such as α-fetoprotein (AFP) are widely used for HCC detection, they still do not provide a satisfactory tool for early detection of HCC. As well as, its sensitivity in detecting small HCC (namely those below 1-2 cm) is rather low. The strongest information on prognosis and possibly driving treatment strategy comes only from histological examination of the tissue, which is not available at the time of diagnosis ⁶. The theory assuming that diabetes mellitus (DM) may increase the risk of HCC has been confirmed in some cohort and case-control studies⁷.

DM was considered as an independent risk factor for hepatocellular carcinoma (HCC), despite the presence of HCV, HBV, alcoholic liver disease, or nonspecific cirrhosis. It was established that DM is associated with a 2–3-fold increase in the risk of HCC development. This finding suggests that it may account for a significant proportion of idiopathic HCC ⁸. Glucose is not reduced by aldose reductase (AR) under normoglycemic conditions, but it follows the normal glycolytic pathway. Increased activity of AR mediates the pathologies associated with DM and is thought to be involved in increased resistance to chemotherapeutic drugs. Thus, the use of AR inhibitors may serve as a protective therapy in cancer^9,10.

AR is the rate-limiting enzyme in the polyol pathway (PP), in which glucose is converted to sorbitol¹¹. It catalyzes NADPH-dependent glucose reduction into sorbitol (sorbitol pathway) and excessive deposition of sorbitol in different tissues of animal models of DM and cell lines cultured in high glucose media which was supposed as an important mediator for the pathogenesis of late diabetic complications ¹². However, it was established that abnormalities of glucose metabolizing enzymes during the transformation of normal hepatocytes necessitate high glucose utilization in hepatoma cells ¹³.

In disrupted intracellular glucokinesis, sorbitol dehydrogenase (SDH) activity, an enzyme responsible for conversion of sorbitol back to fructose ¹⁴ to be utilized better by cells is also disrupted. SDH, the second key enzyme in PP, catalyzes the inter-conversion of polyols, such as sorbitol and xylitol to their respective ketones. SDH deଁciency leads to subsequent accumulation of sorbitol within the cells and diabetic complications ¹⁵. Polyol profile was considered to be an index for early changes during progression to HCC in chemically induced liver cancer. To pursue early pathologic changes, red blood cells (RBCs) were suggested as a short spam vehicle for study investigations¹⁶. The interference of DM with viral hepatitis C (HCV) infection was considered to be a morbidifying risk for hepatocarcinogenesis ¹⁷.

This work aimed to investigate the plasma level of blood hemoglobin, insulin, total protein, glucose, AR, SDH along with the erythrocytic content of total protein, glucose, AR, and SDH. Then correlating these variables to serum AFP, plasma, and RBCs glucose to PP enzyme concentrations, then calculating the insulin/RBCs AR content among the studied groups. Then we will try a mathematical formula from plasma insulin/RBCs AR ratio, hopefully, to find a novel early predictive non-invasive marker for HCC in HCV-infected with or without DM patients. Erythrocytes will be adopted as a possible vehicle for early pre-malignant variations due to their short life span.

Patient groups

In this study, we recruited 120 persons (48–64 years old), classified into 6 equal groups as follows: healthy control, type II DM, HCC, type II DM with HCC, type II DM having HCV infection, and normoglycemic with HCV infection. HCC patients were first grade (without metastasis) and diagnosed by liver biopsy as listed in their hospital files at Heart and Liver Diseases Research Center in Kafrelsheikh, Kafrelsheikh Governorate, Egypt, during 2019-2020. Samples were collected after approval of the Committee of Ethical Guidelines, Kafrelsheikh University, and written informed consents were taken from participating patients by their inspective medical team.

Chemicals

All chemicals were of analytical grade, purchased from Sigma-Aldrich, USA.

Blood samples

Five ml venous blood samples were obtained from each individual after fasting for 8 hours and collected into heparinized tubes. Blood hemoglobin was measured firstly, then all blood samples were subsequently centrifuged at 2000 rpm/min for 10 min then plasma was kept in sterile screw-capped vials at -20ºC right biochemical measurements, the sedimented erythrocytes were washed three times with approximately 2 times its volume cold saline, centrifuged, then the washed RBCs (WRBCs) were kept for biochemical investigations at -5ºC.

Preparation of red blood cell samples for AR and SDH determination

The washed RBCs were transferred into tubes containing 1 ml of acid-citrate-dextrose solution (23 mM citric acid, 45 mM sodium citrate, 82 mM dextrose) and stored at 4 ºC for less than 7 days. No significant change in AR level had been found during 7 days of storage. The samples were mixed well, centrifuged at 1,000 rpm for 10 min, the supernatant was removed and the resultant RBCs were washed twice with 10 vol of ice-cold PBS (137 mM NaCl, 2.7 KCl, 4.3 mM Na₂HP0₄, 1.4 mM KH₂PO₄, pH 7.3), centrifuged for 5 minutes at 2000 rpm and the supernatant was discarded completely. The washed RBCs were frozen at -20˚C¹⁸.

Erythrocytic AR and SDH extraction

Erythrocytes were hemolyzed by adding an equal volume of phosphate buffer (pH 7.0) and by two cycles of freezing and thawing using a dry ice acetone bath. The supernatant fraction of the hemolysate was obtained by centrifugation at 15,000 rpm for 15 min and stored at -80 ºC for erythrocytic AR and SDH determination by ELISA¹⁸.

Extraction of glucose from RBCs

An aliquot of saline-washed frozen RBCs was used for extraction of glucose by adding 2 ml of cold 6% perchloric acid (1.7 ml of pure perchloric acid in 50 ml of distilled water) to 200 μl of washed RBCs. The protein was precipitated and the erythrocytes were centrifuged at 3000 rpm/min at 4°C for 10 min. The supernatant was neutralized at 4°C with 1 M K₂CO3 ¹⁹^,²⁰and then used for measurement of RBCs glucose content.

Extraction of total protein from RBCs

The extraction of total protein was done by haemolysis of RBCs. The cytoplasm of the RBCs contains dissolved salts and sugars. Since the cytoplasm of the RBCs has a higher amount of distilled water, there is a net flow of water into the cell by osmosis. The cell would swell up and eventually bursts and ttherefore the content of total protein in the erythrocytes has been determined. Two ml of distilled water were added to 200 μl of washed RBCs and shacked gently, then centrifuged at 3000 rpm for 15 minutes and the supernatant was used to determine RBCs total protein content ²¹.

Methods

Determination of plasma insulin level

Plasma insulin level was determined by enzyme-linked immunosorbent assay (ELISA) according to the method described before ²², using a commercially available kit from Shanghai Sunred Biological Technology Co., Ltd, China.

Determination of AFP level

Plasma AFP level was determined by ELISA ²³^,²⁴ using a commercially available kit from Cell Biolabs, Inc., San Diego, USA.

Quantitative determination of plasma and RBCs glucose content

This was carried out using a commercial kit obtained from SPINREACT diagnostics, Egypt ²⁵.

Quantitative determination of total protein in plasma and RBCs hemolysate

Total protein was determined according to the method mentioned elsewhere²⁶, using a commercial kit purchased from Elitech Clinical Systems Co, Italy.

Determination of SDH levels in plasm and RBCs

SDH concentration was determined by ELISA ²⁷, using a commercially available kit from Shanghai Sunred Biological Technology Co., Ltd, China

Determination of AR levels in plasm and RBCs

AR was determined by ELISA²⁸using a kit provided from Cloud Clone, USA. Both AR and SDH contents in RBCs were calculated as ng/mg Hb.

Determination of blood Hb% levels

Hemoglobin was measured spectrophotometrically ²⁹ using a commercial kit obtained from SPINREACT diagnostics, Egypt.

Statistical analysis

The data were expressed as Mean ± standard deviation (SD). The difference between groups was determined by using one-way analysis of variance (ANOVA) followed by Tukey^'s-HSD multiple range post hoc test. GraphPad Prism software version 6.0, USA was used for statistical evaluation of the obtained data, and the statistical significance was considered at P < 0.05. Spearman correlation was used to evaluate the associations between different variables.

For each group, plasma insulin was subdivided by erythrocytic AR (bad enzyme) concentration. The resultant insulin/ erythrocytic AR value was the same for both DM and control groups. The insulin/ erythrocytic AR value was then used to figure out a final value that lets us predict progression into HCC in DM or non-treated persistent HCV infected patients. This was done by dividing this figure of DM by the control group which was equal to 1.0. When this figure for the HCV group was divided by the DM group (both are non-HCC-affected), the resultant value was 0.69. This value will be the cut-off value for non-HCC developing individuals. However, this ratio in the HCC group, when divided by both DM or control ratio, the result was the same which was 0.49, taking into account that HCC patients are early, not metastatic groups. Below 0.49 patients will be considered as going to HCC progression.

Thus, this ratio of 0.49 or less is a suggested predictor of HCC development among type II diabetics or non-treated HCV-infected patients.

Blood hemoglobin, plasma, and erythrocytic total protein levelsamong the studied groups

Blood hemoglobin concentration in both HCC and DM with HCC groups was significantly decreased, and this level among HCV and DM with HCV groups was less significantly decreased than both control and DM groups. Plasma and erythrocytic total protein levels in both HCC and DM with HCC groups were significantly decreased, while this level was significantly higher among HCV and DM with HCV groups than both control and DM groups (Table 1).

Plasma insulin and alpha-fetoprotein levelsamong the studied groups

Plasma insulin concentration was significantly increased in DM, HCC, DM with HCC, DM with HCV, and HCV groups than the control group. Plasma AFP concentration in both HCC and DM with HCC groups was significantly increased, and this level was less significantly increased among HCV and DM with HCV groups than both control and DM groups (Table 1).

Plasma and erythrocytic glucose contentamong the studied groups

The level of plasma and erythrocytic glucose among DM, DM with HCC, and DM with HCV groups was significantly increased (P <0.001), compared to healthy control. HCC and HCV-infected groups showed no significant change in blood plasma glucose level when compared to control, being both normoglycemic. But HCC group exhibited a significant decrease in the erythrocytic content of glucose by 30.4% (P <0.01) when compared with the control group (Table 1).

Plasma and erythrocytic content of AR and SDH among the studied groups

The content of plasma and erythrocytic AR in DM with HCV, HCC, and DM with HCC groups was significantly increased (P<0.001) compared to healthy control. As well, HCV-infected and DM groups exhibited a significant increase in the plasma and erythrocytic AR concentration, compared to the control group (Table 1).

Plasma anderythrocytic SDH expression were significantly increased in HCV-infected, HCC, DM with HCC, and DM with HCV groups, compared to healthy control (P<0.001), while in DM, it was significantly decreased (Table 1).

Correlation between AFP and polyol enzymes in plasma and erythrocytes among the studied groups

AFP was significantly and positively correlated with plasma AR (Fig. 1 A, P < 0.0001, r =0.838), and plasma SDH (Fig. 1 B, P < 0.05). As well as, significant positive correlations between plasma AFP and AR content in erythrocytes (Fig.1 C, P < 0.0001),and erythrocytic SDH (Fig. 1 D, P < 0.05) were observed.

The associations of glucose and polyol enzymes in plasma and erythrocytes among the studied groups

Plasma glucose was correlated positively with plasma AR(Fig.1 E, P < 0.01), and negatively with plasma SDH (Fig. 1 F, P < 0.001). Also, there is a significant positive correlation between erythrocytic glucose and erythrocytic AR content (Fig. 1 G, P < 0.01), and a negative correlation with erythrocytic SDH content (Fig. 1 H, P < 0.0001).

Plasma glucose was significantly correlated with erythrocytic AR content (Fig. 1 I, P < 0.0001), while significantly and negatively correlated with erythrocytic SDH content (Fig. 1 J, P < 0.0001). Also, there is a highly significant correlation between plasma glucose level and erythrocytic glucosecontent (Fig. 1 K, P < 0.0001).

The majority of studies conclude that AFP is not a useful diagnostic test for the detection of HCC^30–33, 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 diseases^35,36.

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 development^42–44.

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 mechanisms^47–49.

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 HCC^54,55.

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 metabolism^60,61 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 patients^63,64.

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 stress^67,68.

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 stress^70–72. 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.

Study limitations

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.

AFP is still lacking a satisfactory value for early prediction of the HCC progression among risky subjects as diabetics and persistently non-treated HCV-infected patients. Type II DM is known to cause definite biochemical abnormalities which worsen prognosis and treatment outcomes in HCV-infected patients. Chronic HCV is significantly associated with high expression of AR and SDH in plasma and RBCs.

In our work, HCC was principally associated with disturbed AR and SDH levels in plasma and erythrocytes. AR was significantly increased in DM, HCC, HCC with DM and HCV infected patients, but more elevations were observed in RBCs than plasma. AR activity was higher among cases of HCC with DM than non-diabetic HCC ones.

Plasma and RBCs’ SDH levels were significantly depressed in DM; while these values were significantly elevated in HCC, HCC with DM, HCV+/- DM groups.

The study substantiated the use of the studied RBCs variables as early detection markers for HCC among chronic HCV-infected patients +/- DM, in addition, PP can be advised as a follow-up test among HCV-infected patients and type II diabetics.

The use of insulin/ erythrocytic AR ratio was the same for both DM and control groups. This ratio is useful to predict progression into HCC in DM or non-treated persistent HCV infected patients. The value of 0.69 would be the cut-off value for non-HCC developing individuals. Below 0.49, patients will be considered as going to HCC progression. We recommend the use of insulin/ erythrocytic AR ratio as a non-invasive test, suggested for the first time to predict HCC development among type II diabetics or non-treated HCV-infected patients. Each clinical laboratory will set his own insulin/ erythrocytic AR ratio for normal healthy individuals to be used for calculating the ratio of insulin/ erythrocytic AR ratio for the patient/that of the control.

Funding: None to declare.

Conflicts of interest/Competing interests: Authors declare no conflict of interest.

Availability of data and material: Not applicable.

Ethical approval: This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of University of Zagazig (Date 20 Jan 2017 /No 09). Informed consent was obtained from all individual participants included in the study.

Code availability: Not applicable.

Consent to participate: Done.

Consent for publication: Ok.

Author Contributions

The manuscript was conceptualized by Nabil MA. All authors contributed to the study. Material preparation, data collection and analysis were performed by Asmaa EA, Moustafa SA and Mohamad HS. The first draft of the manuscript was written by Asmaa EA and Nabil MA reviewed, commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Table 1: Variations in blood levels of Hb, alpha fetoprotein, insulin, and both plasma and erythrocyte levels of glucose, total protein, aldose reductase and sorbitol dehydrogenase among studied groups (Values are expressed as mean± SD, N=20):

HCV

Diabetic HCV

Diabetic HCC

HCC

Diabetic

Control

Group

Parameter

13.3 ± 0.4^a,b,c,d

12.6 ± 0.4^a,b,c,d

8.0 ± 0.6^a,b

8.0 ± 0.5^a,b

14.9 ± 0.6

15.2 ± 0.9

Whole blood Hb (g/dl)

9.0± 0.3^a,b,c,d

10.1± 0.3^a,b,c,d

5.7± 0.7^a,b

5.3± 0.7^a,b

7.6±0.5

7.5± 1.1

Plasma total protein (g/dl)

7.5± 0.4^{a, b,c,d}

7.0± 0.4^{a, b, c, d}

3.4± 0.6^a,b

3.7± 0.6^a,b

4.7± 0.2^a

5.5± 0.5

Erythrocyte total protein (g/dl)

16.4± 1.9^b,d,e

28.2± 4.7^a,c

28.6± 4.6^a,c

16.6± 2.2^b

25.5± 6.9^a

13.4± 2.4

Plasma insulin (mU/l)

6.2±1.2^a,c,d

7.2±1.2^c,d

15.0±6.4^a,b

12.8±6.2^a,b

2.4±0.2

2.6 ± 0.3

Plasma AFP (ng/ml)

100.9± 6.7^b,d,e

305.6± 53.3^a,c

287.1± 48.8 ^a,c

69.0± 2.4^b

322.3± 53.0^a

91.8 ± 5.7

Plasma glucose (mg/dl)

19.9±1.5^b,c,d,e

42.6±4.8^a,c,d

36.8±4.9^a,b,c

14.1±0.8^a,b

45.4±5.3^a

20.3 ± 1.4

Erythrocyte glucose (μmol/l)

3.5± 1.7^a,c,d

5.1± 1.4^a,d

9.9± 1.8^a,b,c

6.8± 1.7^a,b

4.4±2.1^a

0.5 ± 0.1

Plasma AR (ng/ml)

7.6± 1.7^{a, c , d,e}

10.1± 2.2^a,c,d

15.2± 2.0^a,b,c

11.5± 1.5^{a, b}

8.1±2.1^a

4.2±0.6

Erythrocyte AR (ng/mg Hb)

12.8±1.2^a,b,c,d,e

8.90± 1.1^a,b,d

5.2± 1.0^b,c

10.3± 1.7^a,b

2.2± 1.7^a

3.9± 0.4

Plasma SDH (ng/ml)

16.0 ±1.2^{a, b,c,d, e}

10.1 ± 1.1^{a,b, c,d}

7.1 ± 1.2^{a, b,c}

13.5 ± 1.4^a,b

1.9 ± 1.9^a

5.1 ± 0.5

Erythrocyte SDH (ng/mg Hb)

Significant difference vs. ^a normal control,^bDiabetic,^c HCC, ^d Diabetic HCC, ^eDiabetic HCV. Significant differences between groups were analyzed by one-way ANOVA test followed by Tukey^'s-HSD multiple range post hoc test, significance was considered at P < 0.05. HCC: Hepatocellular carcinoma, HCV: Hepatitis C virus, AR: Aldose reductase, SDH: Sorbitol dehydrogenase, AFP: Alpha fetoprotein. All diabetic patients were of type II.

No competing interests reported.

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Plasma Insulin/Erythrocytic Aldose Reductase Ratio as a Predictor for Hepatocellular Carcinoma Among Type II Diabetics and Hepatitis C Virus-Infected Patients

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