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Serum vitamin B12 levels as indicators of disease severity and mortality of patients with acute‐on‐chronic liver failure
Clinica Chimica Acta 413 (2012) 1809–1812
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Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Serum vitamin B12 levels as indicators of disease severity and mortality of patients with acute‐on‐chronic liver failure JiuFeng Dou a, WeiYi Xu b, Bo Ye b, Yi Zhang b, WeiLin Mao b,⁎ a b
Department of Traditional Chinese Medicine Pharmacy, First Affiliated Hospital, Zhejiang University School of Medicine, Zhejiang 310003, PR China Department of Clinical Laboratory, First Affiliated Hospital, Zhejiang University School of Medicine, Zhejiang 310003, PR China
a r t i c l e
i n f o
Article history: Received 28 April 2012 Received in revised form 5 July 2012 Accepted 10 July 2012 Available online 16 July 2012 Keywords: Vitamin B12 Acute-on-chronic liver failure (AoCLF) Mortality
a b s t r a c t Background: The study was designed to investigate the association between vitamin B12 levels and patients with acute-on-chronic liver failure (AoCLF). In addition, we analyzed whether B12 is associated with mortality in the AoCLF patients. Methods: One hundred five patients with AoCLF and 44 healthy controls (HCs) were prospectively studied. In all subjects, a blood sample was collected at admission to examine liver function, renal function, international normalized ratio and B12 levels. A total of 9 clinical chemistry and biochemical variables were analyzed for possible association with outcomes by using Cox proportional hazards and multiple regression models. Results: AoCLF patients had significantly higher B12 levels at admission compared with HCs (AoCLF patients vs HCs, median 1218 pg/ml, mean 1736.2 pg/ml, SD, 1294.6 vs 504 pg/ml, 529.8±149.8 pg/ml, Pb 0.001). Elevated B12 levels were associated with increased severity of liver disease and 3-month mortality rate. Multivariate analysis demonstrated that B12 levels and the model for end-stage liver disease score were independent predictors for mortality (both P b 0.001). Conclusion: B12 levels were significantly higher in AoCLF patients than in HCs and elevated B12 were associated with increased severity of disease. Moreover, B12 levels are an independent predicting factor for the 3-month mortality rate in AoCLF patients. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.
1. Introduction Vitamin B12 (cobalamin) is a water-soluble cobalt-containing vitamin. It is an important dietary vitamin required for DNA synthesis, repair and one-carbon metabolism within the cell and the liver plays an important role in the storage and bioavailability of vitamin B12 [1,2]. It has long been noted that circulating concentrations of B12 were abnormally increased in patients with liver disease such as hepatitis, cystic fibrosis, alcoholic cirrhosis, and alcoholic liver diseases [3–6]. For example in acute hepatitis, increased concentrations in plasma have been found in 25 to 40% of the patients [7,8]. Moreover, increased blood vitamin B12 concentration has recently been identified as a prognostic indicator for patients with hepatocellular carcinoma [9]. Liver failure is an important cause of death worldwide. In China, hepatitis B virus (HBV)-related acute-on-chronic liver failure (HBV-related AoCLF) is the most common type of liver failure. It occurs because of sudden and severe impairment or loss of hepatocyte functions in patients with chronic liver diseases [10]. AoCLF has been shown to carry poor
⁎ Corresponding author at: Department of Clinical Laboratory, First Affiliated Hospital, Zhejiang University School of Medicine, #79 Qingchun Road, Hangzhou, Zhejiang Province, PR China, 310003. Tel.: +86 571 87236382; fax: +86 571 87236383. E-mail address: [email protected] (W. Mao).
prognosis, with an in-hospital mortality rate of >70% if liver transplantation is not available [11–13]. However, to our knowledge, serum B12 concentrations in patients with AoCLF have not been well-defined. We hypothesized that B12 concentrations would be possibly associated with clinical outcomes in AoCLF patients. Therefore, we conducted a prospective study to evaluate the prognostic value of B12 in AoCLF patients. 2. Materials and methods 2.1. Subjects A total of 105 consecutive patients with AoCLF formed the AoCLF group, ranging in age from 31 to 66 y (mean ± SD, 47.2 ± 11.2 y; 29 women and 76 men), whereas 44 healthy controls (HCs) formed the HC group, ranging in age from 32 to 61 y (mean ± SD, 45.3± 10.9 y; 10 women and 34 men). The study was made between April 2010 and September 2011 in our hospital (First Affiliated Hospital of Zhejiang University School of Medicine). In the present study, patients who received anti-HBV agent or steroid 6-month before admission were excluded. Patients with a concurrent infection of HCV, hepatitis D virus, hepatitis G virus, and/or human immunodeficiency virus and any autoimmune liver disease were also excluded. We took a detailed history of all the patients and we included only the patients who never took
0009-8981/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2012.07.008
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alcohol. HCs were recruited from among community-dwelling individuals who presented for their yearly physical examinations and had no specific complaints or illness requiring treatment. The diagnosis of HBV-related AoCLF was according to the guidelines and recommendations of the Asian Pacific Association for the Study of the Liver [14]. Briefly, AoCLF is defined as acute hepatic insult manifesting as jaundice and coagulopathy, complicated within 4 weeks by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease. In addition, a diagnosis of hepatic encephalopathy was made based on the clinical manifestations and signs of brain edema as determined using CT or magnetic resonance imaging. All selected patients received AoCLF-modifying standard medical treatments at the hospital. This included absolute bed rest; intravenous drip infusion of albumin and plasma; attention to maintain water, electrolyte and acid–base balance. The survival length of 105 AoCLF patients was observed for at least 4 months. Patient follow-up was performed by means of telephone calls, personal interviews, and review of clinical reports. Date of death was obtained from scrutiny of medical records. The study was approved by the ethics committee of the First Affiliated Hospital of Zhejiang University School of Medicine, and written informed consent for participation was obtained from each study participant. 2.2. Laboratory analysis Blood samples were collected from all AoCLF patients within 24 h after admission, and blood samples were taken from 44 healthy individuals at the time of recruitment. Serum vitamin B12 concentration was measured using an Immulite® 2000 analyzer (Diagnostic Products Corp., Los Angeles, CA) with the chemiluminescence method. Reference range for vitamin B12 is 240–1200 pg/ml. Serum creatinine, serum albumin, total protein, total bilirubin and alanine aminotransferase (ALT) activities were measured using the Hitachi 704 Analyzer (Roche Diagnostics, Indianapolis, IN), international normalized ratio (INR) was done by Sysmex CA1500 full-automatic analyzer (Sysmex Corp, Hyogo, Japan). At baseline, demographic and clinical characteristics, including the model for end-stage liver disease (MELD) score (with higher scores indicating more severe illness), were collected. 2.3. MELD score Liver disease severity was evaluated via MELD score, which uses the patient's serum bilirubin and creatinine concentrations and the INR for prothrombin time to predict survival. The MELD score was calculated using the web site calculator (http://www.mayoclinic. org/gi-rst/mayomodel7.html).
3. Results 3.1. Increased serum vitamin B12 concentrations in AoCLF patients Serum vitamin B12 concentrations in AoCLF patients have not been described previously. No correlation between the B12 concentration and age was observed in HC group (r = 0.03, P =0.59) and AoCLF group (r = 0.08, P = 0.38). Our study showed that the B12 concentrations in AoCLF patients were significantly higher than HCs (AoCLF patients vs HCs, median 1218 pg/ml, mean 1736.2 pg/ml, SD, 1294.6 vs 504 pg/ml, 529.8 ± 149.8 pg/ml, P b 0.001) (Fig. 1). 3.2. Baseline characteristics and baseline factors related with vitamin B12 concentrations A total of 105 participants were eligible for this study. Patients were divided into three groups based on their B12 concentrations, group A (B12 ≤1200), group B (>1200, but b 2000) and group C (≥2000). Clinical and laboratory characteristics among patients with different B12 concentrations are listed in Table 1. Patients with in the higher B12 concentrations were more likely to have severe liver disease, had lower concentrations of serum albumin, and had higher INR, ALT and total bilirubin. Moreover, highly increased serum B12 concentration was associated with a higher rate of clinical complications such as cirrhosis and ascites. The serum creatinine, gender and total protein were not significantly different among the three groups. The MELD score in groups A, B, and C was 11.1 ± 4.4, 17.2 ± 4.7 and 20.5± 5.1, respectively. There was a stepwise increase in MELD scores with increasing B12 concentrations (P b 0.001 between groups A and B, and P b 0.01 between groups B and C) (Fig. 2, left). 3.3. Association of B12 concentrations with 3-month mortality in AoCLF patients The median follow-up period was 81 days (range, 24–121 days). During the follow-up, 34 patients died within 3 months. There was a significant increase in 3-month mortality rate following increasing B12 concentrations, with 13.3% in group A, 36.7% in group B and 70.8% in group C (Pb 0.01 between groups A and B, and between groups B and C) (Fig. 2, middle). Kaplan–Meier analysis showed a graded increased probability of death with increasing B12 concentrations (log-rank Pb 0.01) (Fig. 2, left). To evaluate the value for B12 concentrations and MELD score to predict mortality, a ROC curve was drawn (Fig. 3). The AUC was calculated as 0.815±0.044 for MELD score and 0.659±0.054 for B12 concentrations
2.4. Statistical analysis All continuous variables were expressed as mean value ± standard deviation (SD), and categorical data as percentages. We used SPSS ver 15.0 (SPSS, Inc., Chicago, IL) to perform the statistical procedures. The Kruskal–Wallis H test and Mann–Whitney nonparametric U test were used for comparison between groups. Categorical data were evaluated by the χ 2-test or Fisher's exact test, as appropriate. Correlations between variables were examined with a Spearman correlation. The log-rank test was used to test for differences in survival between groups. Cox proportional hazards models were used to evaluate the association between each baseline variable and all-cause death. A multivariable stepwise logistic regression test was used to evaluate the independent clinical parameters predicting mortality. The receiver operating characteristic (ROC) curve was obtained and area under the curve (AUC) was calculated to identify the best B12 to predict mortality in patients with AoCLF. A P b 0.05 was considered statistically significant.
Fig. 1. The serum B12 levels in patients with AoCLF and healthy controls at admission. Data are expressed as box plots, in which the horizontal lines illustrate the 25th, 50th, and 75th percentiles of the B12 levels. The vertical lines represent the 5th and 95th percentiles.
J. Dou et al. / Clinica Chimica Acta 413 (2012) 1809–1812
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Table 1 Clinical and laboratory characteristics among patients with different vitamin B12 levels (pg/ml) at admission.
B12 (pg/ml)a Age (year)a Gender (male/female) Total protein (g/L)a Albumin (g/L)a ALT (U/L)a INRa Creatinine (mmol/L)a Total bilirubin (μmol/L)a MELD scorea Hepatic cirrhosis (yes/no) Ascites (yes/no) Mortality (yes/no)
Group A (B12 ≤1200, n= 51)
Group B (1200 b B12 b 2000, n = 30)
Group C (B12 ≥2000, n= 24)
P
821.8 ± 142.2 42.9 ± 12.5 39/8 63.0 ± 6.8 37.8 ± 5.9 163.1 ± 355.1 1.32 ± 0.36 64.3 ± 12.2 93.6 ± 88.5 10.7 ± 4.1 5/46 2/49 6/45
1537.1 ± 282.7 49.4 ± 13.1 44/9 61.5 ± 7.7 34.7 ± 5.4 209.6 ± 279.5 1.64 ± 0.44 67.2 ± 27.8 214.7 ± 163.5 15.3 ± 4.7 11/19 7/23 11/19
3643.0 ± 873.3 47.0 ± 12.7 16/7 59.0 ± 7.9 33.0 ± 4.9 275.1 ± 786.5 2.29 ± 1.13 85.3 ± 43.5 264.1 ± 191.8 19.8 ± 4.4 20/4 19/5 17/7
b0.001 0.059 0.077 0.123 0.024 0.025 b0.001 0.914 0.001 b0.001 b0.001 b0.001 b0.01
Abbreviations: INR, international normalized ratio; ALT, alanine aminotransferase; MELD score, model for end-stage liver disease score. a Data were expressed as mean ± standard deviation.
(both P b 0.001). When B12 and MELD were combined, the AUC was 0.930 ± 0.024 (P b 0.001). Univariate logistic regression analysis demonstrated that those patients with higher B12 concentrations, higher MELD score, higher ALT, higher creatinine, higher total bilirubin, lower total protein and lower albumin concentration had significantly greater death hazard. The multivariate logistic regression analysis showed that only B12 concentrations and MELD score were independent factors predicting mortality rate (Table 2). 4. Discussion Because the liver plays an essential role in the storage of vitamin B12, it is not amazing that liver diseases are associated with major changes in serum vitamin B12 concentrations. Cylwik et al. [15] reported that the vitamin B12 concentrations in patients of alcohol abuse are significantly higher than those of healthy subjects, despite normal serum vitamin B12 concentrations. Fragasso et al. [16] reported that the serum vitamin B12 concentrations may decrease in some alcohol-dependent patients with megaloblastic anemia, although these still remain in the reference range. Ermens et al. [2] reported that in liver cirrhosis, plasma vitamin B12 concentrations can reach high values depending on the severity of the disease. However, Holdsworth et al. [17] reported that a significant number of cirrhotic patients have lower serum vitamin B12 concentration in their study. In our study, patients with AoCLF had significantly higher B12 concentrations compared with healthy subjects. It is not clear why AoCLF patients showed increased blood B12. Indeed, high B12 concentrations of AoCLF patients were shown to correlate with liver dysfunction determined by increased concentrations of ALT (Table 1). It has been hypothesized that inflammation-induced hepatocyte degradation in virus infection and/or pathological destruction of hepatocytes in liver cirrhosis causes the release of cobalamin from the liver. Several studies have
shown that increased plasma cobalamin was associated with tissue B12 depletion in liver cirrhosis [18,19]. The total B12 content of livers from hepatoma-bearing rats was less than that in control animals [20]. Our observations taken together with the other's findings suggest that liver injuries may in part contribute to increased B12 concentrations of AoCLF patients. The most interesting finding from our study is that increasing B12 concentrations can serve as an independent predictor of mortality in patients with AoCLF (Fig. 3 and Table 2). Over the past decade, the MELD score is the method most widely used for organ allocation in liver transplantation. This model, which includes variables related to both liver and renal functions, was implemented in the USA in 2002 and is currently being used in many countries to classify patients with cirrhosis awaiting transplantation according to the severity of their liver disease [21]. Our previous study reported that the model for MELD score was related to the prognosis of the patients with hepatitis B virus (HBV)-related acute-on-chronic liver failure (AoCLF) [22]. In the present study, we reported that vitamin B12 can be used for predicting HBV-infected patients' mortality, although the prediction power of B12 was relatively lower (AUC = 0.664± 0.049, P b 0.001) than that of MELD score (AUC = 0.847± 0.034, P b 0.001). Moreover, combining B12 concentrations with MELD score further added to prediction power of predicting mortality (AUC= 0.930± 0.024, P b 0.001). Patients with high MELD score and high B12 concentrations carry the highest risk of mortality. The mechanism underlying the association between B12 concentrations and death in AoCLF patients is unclear. Indeed, inflammation in HBV-infected liver is proven to be mediated by cytokines, which have been claimed to play a pivotal role in the pathogenesis of chronic HBV infection [23,24]. A number of cytokines are released from macrophages or monocytes in response to stimulation by endotoxins, and these cytokines affect disease status [25]. Consistently, we also found an association between B12 concentrations and the
Fig. 2. Comparisons of the model for end-stage liver disease (MELD) score (left), mortality rate (middle) and survival curves (right) among patients with different serum B12 levels. Patients were divided into three groups based on serum RDW values, group A (B12 ≤1200), group B (>1200, but b2000) and group C (≥2000).
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J. Dou et al. / Clinica Chimica Acta 413 (2012) 1809–1812
Abbreviations HBV hepatitis B virus HCs healthy controls AoCLF acute-on-chronic liver failure MELD score model for end-stage liver disease score
Acknowledgment The work was supported by grants from Health Department of Zhejiang Province (2011KYA058). WeiLin Mao proposed the study. JiuFeng Dou performed research and WeiYi Xu wrote the first draft. Bo Ye and Yi Zhang collected and analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. WeiLin Mao is the guarantor. References
Fig. 3. Receiver operating characteristic (ROC) curve analysis for prediction of mortality by serum B12 levels (‐‐‐ line), model for end-stage liver disease (MELD) scores (–– line) and their combination (⋯ line) at admission.
severity of the liver disease, patients with higher B12 concentrations had lower concentrations of serum albumin, and had higher INR, ALT and total bilirubin. Moreover, highly increased serum B12 concentration was associated with a higher rate of clinical complications such as cirrhosis and ascites (Table 1). We hypothesized that the association of B12 concentrations with mortality risk may, in part, be due to an effect of inflammation on liver and risk. A few limitations warrant consideration. Firstly, this was a singlecenter study and our sample size may have posed a limitation of this study. The findings need to be confirmed in multi-center and prospectively designed studies. Secondly, vitamin B12 was not dynamically observed, and thus, whether RDW values are stepwise increased when patient's condition is progressively deteriorated remains unclear. In conclusion, the study confirms a high prevalence of abnormally increased serum B12 concentrations in AoCLF patients. The determinants of increased serum B12 concentrations were in association with liver injuries. Our data identify a poor prognostic influence of increased serum B12 concentrations in AoCLF patients. The molecular marker of increased B12 concentrations adds predictive value to the conventional clinical factors such as MELD score, which in combination may be prognostically useful for early detection of disease progression and better treatment strategies.
Table 2 Cox proportional hazards analysis for predictors of death. Univariable
B12 (pg/ml) MELD score Age (y) Total protein (g/L) Albumin (g/L) ALT (U/L) Creatinine (mmol/L) Total bilirubin (μmol/L)
Multivariable
Odds ratio
95% CI
P
Odds ratio
95% CI
P
1.980 2.889 1.033 0.894 0.827 1.106 1.018 1.008
1.485–2.159 2.288–3.728 1.000–1.007 0.829–0.965 0.748–0.914 1.097–1.201 1.018–1.072 1.005–1.012
b0.001 b0.001 0.053 0.004 b0.001 0.013 0.001 b0.001
1.891 2.512 – – – – – –
1.329–2.049 1.977–3.153 – – – – – –
0.021 0.005 NS NS NS NS NS NS
Abbreviations: MELD score, model for end-stage liver disease score.
[1] Bailey SW, Ayling JE. The extremely slow and variable activity of dihydrofolate reductase in human liver and its implications for high folic acid intake. Proc Natl Acad Sci 2009;106:15424-9. [2] Ermens AA, Vlasveld LT, Lindemans J. Significance of elevated cobalamin (vitamin B12) concentrations in blood. Clin Biochem 2003;36:585-90. [3] Lildballe DL, Nguyen KQ, Poulsen SS, Nielsen HO, Nexo E. Haptocorrin as marker of disease progression in fibrolamellar hepatocellular carcinoma. Eur J Surg Oncol 2011;37:72-9. [4] Baker H, Frank O, DeAngelis B. Plasma vitamin B12 titres as indicators of disease severity and mortality of patients with alcoholic hepatitis. Alcohol 1987;22:1-5. [5] Lindemans J, Abels J, Neijens HJ, Kerrebijn KF. Elevated vitamin B12 in cystic fibrosis. Acta Paediatr Scand 1984;73:768-71. [6] Majumdar SK, Shaw GK, O'Gorman P, et al. Blood vitamin status (B1, B2, B6, folic acid and B12) in patients with alcoholic liver disease. Int J Vitam Nutr Res 1982;52:266-71. [7] Areekul S, Panatampon P, Doungbarn J. Vitamin B12 and vitamin B12 binding proteins in liver disease. Southeast Asian J Trop Med Public Health 1977;8:322-8. [8] Hagelskjaer L, Rasmussen K. Methylmalonic acid concentration in serum not affected in hepatic disease. Clin Chem 1992;38:493-5. [9] Lin CY, Kuo CS, Lu CL, Wu MY, Huang RF. Elevated serum vitamin B12 concentrations in association with tumor markers as the prognostic factors predictive for poor survival in patients with hepatocellular carcinoma. Nutr Cancer 2010;62:190-7. [10] Jalan R, Williams R. Acute-on-chronic liver failure: pathophysiological basis of therapeutic options. Blood Purif 2002;20:252-61. [11] Marrero J, Martinez FJ, Hyzy R. Advances in critical care hepatology. Am J Respir Crit Care Med 2003;168:1421-6. [12] Katoonizadeh A, Laleman W, Verslype C, et al. Early features of acute-on-chronic alcoholic liver failure: a prospective cohort study. Gut 2010;59:1561-9. [13] Chen LG, Guleng B, Ren JL, et al. Artificial liver support system in treatment of liver failure after acute poisoning. World J Emerg Med 2011;4:283-6. [14] Sarin SK, Kumar A, Almeida JA, et al. Acute-on-chronic liver failure: consensus recommendations of the Asian Pacific Association for the Study of the Liver (APASL). Hepatol Int 2009;3:269-82. [15] Cylwik B, Czygier M, Daniluk M, Chrostek L, Szmitkowski M. Vitamin B12 concentration in the blood of alcoholics. Pol Merkur Lekarski 2010;28:122-5. [16] Fragasso A, Mannarella C, Ciancio A, Sacco A. Functional vitamin B12 deficiency in alcoholics: an intriguing finding in a retrospective study of megaloblastic anemic patients. Eur J Intern Med 2010;21:97–100. [17] Holdsworth CD, Atkinson M, Dossett JA, Hall R. An assessment of the diagnostic and prognostic value of serum vitamin B12 concentrations in liver disease. Gut 1964;5:601-6. [18] Djalali M, Champigneulle B, Guéant JL, et al. Increased serum corrinoids correlates with disease severity and IgA concentrations in alcoholic cirrhosis. Digestion 1988;41:215-22. [19] Kanazawa S, Herbert V. Total corrinoid, cobalamin and cobalamin analogue concentrations may be normal in serum despite cobalamin in liver depletion in patients with alcoholism. Lab Invest 1985;53:108-10. [20] Linnell JC, Quadros EV, Matthews DM, Morris HP, Poirier LA. Altered cobalamin distribution in rat hepatomas and in the livers of rats treated with diethylnitrosamine. Cancer Res 1977;37:2975-8. [21] Freeman Jr RB, Wiesner RH, Harper A, et al. The new liver allocation system: moving toward evidence-based transplantation policy. Liver Transpl 2002;8:851-8. [22] Mao W, Ye B, Lin S, et al. The prediction value of MELD scoring system on prognosis in the acute on chronic liver failure patients with artificial liver support system. ASAIO J 2010;56:475-8. [23] Chisari FV, Ferrari C. Hepatitis B virus immunopathogenesis. Annu Rev Immunol 1995;13:29-60. [24] Bertoletti A, D'Elios MM, Boni C, et al. Different cytokine profiles of intrahepatic T cells in chronic hepatitis B and hepatitis C virus infections. Gastroenterology 1997;112:193-9. [25] Mao WL, Chen Y, Chen YM, Li LJ. Changes of serum cytokine concentrations in patients with acute on chronic liver failure treated by artificial liver support system. J Clin Gastroenterol 2011;45:551-5.
Contents lists available at SciVerse ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Serum vitamin B12 levels as indicators of disease severity and mortality of patients with acute‐on‐chronic liver failure JiuFeng Dou a, WeiYi Xu b, Bo Ye b, Yi Zhang b, WeiLin Mao b,⁎ a b
Department of Traditional Chinese Medicine Pharmacy, First Affiliated Hospital, Zhejiang University School of Medicine, Zhejiang 310003, PR China Department of Clinical Laboratory, First Affiliated Hospital, Zhejiang University School of Medicine, Zhejiang 310003, PR China
a r t i c l e
i n f o
Article history: Received 28 April 2012 Received in revised form 5 July 2012 Accepted 10 July 2012 Available online 16 July 2012 Keywords: Vitamin B12 Acute-on-chronic liver failure (AoCLF) Mortality
a b s t r a c t Background: The study was designed to investigate the association between vitamin B12 levels and patients with acute-on-chronic liver failure (AoCLF). In addition, we analyzed whether B12 is associated with mortality in the AoCLF patients. Methods: One hundred five patients with AoCLF and 44 healthy controls (HCs) were prospectively studied. In all subjects, a blood sample was collected at admission to examine liver function, renal function, international normalized ratio and B12 levels. A total of 9 clinical chemistry and biochemical variables were analyzed for possible association with outcomes by using Cox proportional hazards and multiple regression models. Results: AoCLF patients had significantly higher B12 levels at admission compared with HCs (AoCLF patients vs HCs, median 1218 pg/ml, mean 1736.2 pg/ml, SD, 1294.6 vs 504 pg/ml, 529.8±149.8 pg/ml, Pb 0.001). Elevated B12 levels were associated with increased severity of liver disease and 3-month mortality rate. Multivariate analysis demonstrated that B12 levels and the model for end-stage liver disease score were independent predictors for mortality (both P b 0.001). Conclusion: B12 levels were significantly higher in AoCLF patients than in HCs and elevated B12 were associated with increased severity of disease. Moreover, B12 levels are an independent predicting factor for the 3-month mortality rate in AoCLF patients. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.
1. Introduction Vitamin B12 (cobalamin) is a water-soluble cobalt-containing vitamin. It is an important dietary vitamin required for DNA synthesis, repair and one-carbon metabolism within the cell and the liver plays an important role in the storage and bioavailability of vitamin B12 [1,2]. It has long been noted that circulating concentrations of B12 were abnormally increased in patients with liver disease such as hepatitis, cystic fibrosis, alcoholic cirrhosis, and alcoholic liver diseases [3–6]. For example in acute hepatitis, increased concentrations in plasma have been found in 25 to 40% of the patients [7,8]. Moreover, increased blood vitamin B12 concentration has recently been identified as a prognostic indicator for patients with hepatocellular carcinoma [9]. Liver failure is an important cause of death worldwide. In China, hepatitis B virus (HBV)-related acute-on-chronic liver failure (HBV-related AoCLF) is the most common type of liver failure. It occurs because of sudden and severe impairment or loss of hepatocyte functions in patients with chronic liver diseases [10]. AoCLF has been shown to carry poor
⁎ Corresponding author at: Department of Clinical Laboratory, First Affiliated Hospital, Zhejiang University School of Medicine, #79 Qingchun Road, Hangzhou, Zhejiang Province, PR China, 310003. Tel.: +86 571 87236382; fax: +86 571 87236383. E-mail address: [email protected] (W. Mao).
prognosis, with an in-hospital mortality rate of >70% if liver transplantation is not available [11–13]. However, to our knowledge, serum B12 concentrations in patients with AoCLF have not been well-defined. We hypothesized that B12 concentrations would be possibly associated with clinical outcomes in AoCLF patients. Therefore, we conducted a prospective study to evaluate the prognostic value of B12 in AoCLF patients. 2. Materials and methods 2.1. Subjects A total of 105 consecutive patients with AoCLF formed the AoCLF group, ranging in age from 31 to 66 y (mean ± SD, 47.2 ± 11.2 y; 29 women and 76 men), whereas 44 healthy controls (HCs) formed the HC group, ranging in age from 32 to 61 y (mean ± SD, 45.3± 10.9 y; 10 women and 34 men). The study was made between April 2010 and September 2011 in our hospital (First Affiliated Hospital of Zhejiang University School of Medicine). In the present study, patients who received anti-HBV agent or steroid 6-month before admission were excluded. Patients with a concurrent infection of HCV, hepatitis D virus, hepatitis G virus, and/or human immunodeficiency virus and any autoimmune liver disease were also excluded. We took a detailed history of all the patients and we included only the patients who never took
0009-8981/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2012.07.008
1810
J. Dou et al. / Clinica Chimica Acta 413 (2012) 1809–1812
alcohol. HCs were recruited from among community-dwelling individuals who presented for their yearly physical examinations and had no specific complaints or illness requiring treatment. The diagnosis of HBV-related AoCLF was according to the guidelines and recommendations of the Asian Pacific Association for the Study of the Liver [14]. Briefly, AoCLF is defined as acute hepatic insult manifesting as jaundice and coagulopathy, complicated within 4 weeks by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease. In addition, a diagnosis of hepatic encephalopathy was made based on the clinical manifestations and signs of brain edema as determined using CT or magnetic resonance imaging. All selected patients received AoCLF-modifying standard medical treatments at the hospital. This included absolute bed rest; intravenous drip infusion of albumin and plasma; attention to maintain water, electrolyte and acid–base balance. The survival length of 105 AoCLF patients was observed for at least 4 months. Patient follow-up was performed by means of telephone calls, personal interviews, and review of clinical reports. Date of death was obtained from scrutiny of medical records. The study was approved by the ethics committee of the First Affiliated Hospital of Zhejiang University School of Medicine, and written informed consent for participation was obtained from each study participant. 2.2. Laboratory analysis Blood samples were collected from all AoCLF patients within 24 h after admission, and blood samples were taken from 44 healthy individuals at the time of recruitment. Serum vitamin B12 concentration was measured using an Immulite® 2000 analyzer (Diagnostic Products Corp., Los Angeles, CA) with the chemiluminescence method. Reference range for vitamin B12 is 240–1200 pg/ml. Serum creatinine, serum albumin, total protein, total bilirubin and alanine aminotransferase (ALT) activities were measured using the Hitachi 704 Analyzer (Roche Diagnostics, Indianapolis, IN), international normalized ratio (INR) was done by Sysmex CA1500 full-automatic analyzer (Sysmex Corp, Hyogo, Japan). At baseline, demographic and clinical characteristics, including the model for end-stage liver disease (MELD) score (with higher scores indicating more severe illness), were collected. 2.3. MELD score Liver disease severity was evaluated via MELD score, which uses the patient's serum bilirubin and creatinine concentrations and the INR for prothrombin time to predict survival. The MELD score was calculated using the web site calculator (http://www.mayoclinic. org/gi-rst/mayomodel7.html).
3. Results 3.1. Increased serum vitamin B12 concentrations in AoCLF patients Serum vitamin B12 concentrations in AoCLF patients have not been described previously. No correlation between the B12 concentration and age was observed in HC group (r = 0.03, P =0.59) and AoCLF group (r = 0.08, P = 0.38). Our study showed that the B12 concentrations in AoCLF patients were significantly higher than HCs (AoCLF patients vs HCs, median 1218 pg/ml, mean 1736.2 pg/ml, SD, 1294.6 vs 504 pg/ml, 529.8 ± 149.8 pg/ml, P b 0.001) (Fig. 1). 3.2. Baseline characteristics and baseline factors related with vitamin B12 concentrations A total of 105 participants were eligible for this study. Patients were divided into three groups based on their B12 concentrations, group A (B12 ≤1200), group B (>1200, but b 2000) and group C (≥2000). Clinical and laboratory characteristics among patients with different B12 concentrations are listed in Table 1. Patients with in the higher B12 concentrations were more likely to have severe liver disease, had lower concentrations of serum albumin, and had higher INR, ALT and total bilirubin. Moreover, highly increased serum B12 concentration was associated with a higher rate of clinical complications such as cirrhosis and ascites. The serum creatinine, gender and total protein were not significantly different among the three groups. The MELD score in groups A, B, and C was 11.1 ± 4.4, 17.2 ± 4.7 and 20.5± 5.1, respectively. There was a stepwise increase in MELD scores with increasing B12 concentrations (P b 0.001 between groups A and B, and P b 0.01 between groups B and C) (Fig. 2, left). 3.3. Association of B12 concentrations with 3-month mortality in AoCLF patients The median follow-up period was 81 days (range, 24–121 days). During the follow-up, 34 patients died within 3 months. There was a significant increase in 3-month mortality rate following increasing B12 concentrations, with 13.3% in group A, 36.7% in group B and 70.8% in group C (Pb 0.01 between groups A and B, and between groups B and C) (Fig. 2, middle). Kaplan–Meier analysis showed a graded increased probability of death with increasing B12 concentrations (log-rank Pb 0.01) (Fig. 2, left). To evaluate the value for B12 concentrations and MELD score to predict mortality, a ROC curve was drawn (Fig. 3). The AUC was calculated as 0.815±0.044 for MELD score and 0.659±0.054 for B12 concentrations
2.4. Statistical analysis All continuous variables were expressed as mean value ± standard deviation (SD), and categorical data as percentages. We used SPSS ver 15.0 (SPSS, Inc., Chicago, IL) to perform the statistical procedures. The Kruskal–Wallis H test and Mann–Whitney nonparametric U test were used for comparison between groups. Categorical data were evaluated by the χ 2-test or Fisher's exact test, as appropriate. Correlations between variables were examined with a Spearman correlation. The log-rank test was used to test for differences in survival between groups. Cox proportional hazards models were used to evaluate the association between each baseline variable and all-cause death. A multivariable stepwise logistic regression test was used to evaluate the independent clinical parameters predicting mortality. The receiver operating characteristic (ROC) curve was obtained and area under the curve (AUC) was calculated to identify the best B12 to predict mortality in patients with AoCLF. A P b 0.05 was considered statistically significant.
Fig. 1. The serum B12 levels in patients with AoCLF and healthy controls at admission. Data are expressed as box plots, in which the horizontal lines illustrate the 25th, 50th, and 75th percentiles of the B12 levels. The vertical lines represent the 5th and 95th percentiles.
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Table 1 Clinical and laboratory characteristics among patients with different vitamin B12 levels (pg/ml) at admission.
B12 (pg/ml)a Age (year)a Gender (male/female) Total protein (g/L)a Albumin (g/L)a ALT (U/L)a INRa Creatinine (mmol/L)a Total bilirubin (μmol/L)a MELD scorea Hepatic cirrhosis (yes/no) Ascites (yes/no) Mortality (yes/no)
Group A (B12 ≤1200, n= 51)
Group B (1200 b B12 b 2000, n = 30)
Group C (B12 ≥2000, n= 24)
P
821.8 ± 142.2 42.9 ± 12.5 39/8 63.0 ± 6.8 37.8 ± 5.9 163.1 ± 355.1 1.32 ± 0.36 64.3 ± 12.2 93.6 ± 88.5 10.7 ± 4.1 5/46 2/49 6/45
1537.1 ± 282.7 49.4 ± 13.1 44/9 61.5 ± 7.7 34.7 ± 5.4 209.6 ± 279.5 1.64 ± 0.44 67.2 ± 27.8 214.7 ± 163.5 15.3 ± 4.7 11/19 7/23 11/19
3643.0 ± 873.3 47.0 ± 12.7 16/7 59.0 ± 7.9 33.0 ± 4.9 275.1 ± 786.5 2.29 ± 1.13 85.3 ± 43.5 264.1 ± 191.8 19.8 ± 4.4 20/4 19/5 17/7
b0.001 0.059 0.077 0.123 0.024 0.025 b0.001 0.914 0.001 b0.001 b0.001 b0.001 b0.01
Abbreviations: INR, international normalized ratio; ALT, alanine aminotransferase; MELD score, model for end-stage liver disease score. a Data were expressed as mean ± standard deviation.
(both P b 0.001). When B12 and MELD were combined, the AUC was 0.930 ± 0.024 (P b 0.001). Univariate logistic regression analysis demonstrated that those patients with higher B12 concentrations, higher MELD score, higher ALT, higher creatinine, higher total bilirubin, lower total protein and lower albumin concentration had significantly greater death hazard. The multivariate logistic regression analysis showed that only B12 concentrations and MELD score were independent factors predicting mortality rate (Table 2). 4. Discussion Because the liver plays an essential role in the storage of vitamin B12, it is not amazing that liver diseases are associated with major changes in serum vitamin B12 concentrations. Cylwik et al. [15] reported that the vitamin B12 concentrations in patients of alcohol abuse are significantly higher than those of healthy subjects, despite normal serum vitamin B12 concentrations. Fragasso et al. [16] reported that the serum vitamin B12 concentrations may decrease in some alcohol-dependent patients with megaloblastic anemia, although these still remain in the reference range. Ermens et al. [2] reported that in liver cirrhosis, plasma vitamin B12 concentrations can reach high values depending on the severity of the disease. However, Holdsworth et al. [17] reported that a significant number of cirrhotic patients have lower serum vitamin B12 concentration in their study. In our study, patients with AoCLF had significantly higher B12 concentrations compared with healthy subjects. It is not clear why AoCLF patients showed increased blood B12. Indeed, high B12 concentrations of AoCLF patients were shown to correlate with liver dysfunction determined by increased concentrations of ALT (Table 1). It has been hypothesized that inflammation-induced hepatocyte degradation in virus infection and/or pathological destruction of hepatocytes in liver cirrhosis causes the release of cobalamin from the liver. Several studies have
shown that increased plasma cobalamin was associated with tissue B12 depletion in liver cirrhosis [18,19]. The total B12 content of livers from hepatoma-bearing rats was less than that in control animals [20]. Our observations taken together with the other's findings suggest that liver injuries may in part contribute to increased B12 concentrations of AoCLF patients. The most interesting finding from our study is that increasing B12 concentrations can serve as an independent predictor of mortality in patients with AoCLF (Fig. 3 and Table 2). Over the past decade, the MELD score is the method most widely used for organ allocation in liver transplantation. This model, which includes variables related to both liver and renal functions, was implemented in the USA in 2002 and is currently being used in many countries to classify patients with cirrhosis awaiting transplantation according to the severity of their liver disease [21]. Our previous study reported that the model for MELD score was related to the prognosis of the patients with hepatitis B virus (HBV)-related acute-on-chronic liver failure (AoCLF) [22]. In the present study, we reported that vitamin B12 can be used for predicting HBV-infected patients' mortality, although the prediction power of B12 was relatively lower (AUC = 0.664± 0.049, P b 0.001) than that of MELD score (AUC = 0.847± 0.034, P b 0.001). Moreover, combining B12 concentrations with MELD score further added to prediction power of predicting mortality (AUC= 0.930± 0.024, P b 0.001). Patients with high MELD score and high B12 concentrations carry the highest risk of mortality. The mechanism underlying the association between B12 concentrations and death in AoCLF patients is unclear. Indeed, inflammation in HBV-infected liver is proven to be mediated by cytokines, which have been claimed to play a pivotal role in the pathogenesis of chronic HBV infection [23,24]. A number of cytokines are released from macrophages or monocytes in response to stimulation by endotoxins, and these cytokines affect disease status [25]. Consistently, we also found an association between B12 concentrations and the
Fig. 2. Comparisons of the model for end-stage liver disease (MELD) score (left), mortality rate (middle) and survival curves (right) among patients with different serum B12 levels. Patients were divided into three groups based on serum RDW values, group A (B12 ≤1200), group B (>1200, but b2000) and group C (≥2000).
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Abbreviations HBV hepatitis B virus HCs healthy controls AoCLF acute-on-chronic liver failure MELD score model for end-stage liver disease score
Acknowledgment The work was supported by grants from Health Department of Zhejiang Province (2011KYA058). WeiLin Mao proposed the study. JiuFeng Dou performed research and WeiYi Xu wrote the first draft. Bo Ye and Yi Zhang collected and analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. WeiLin Mao is the guarantor. References
Fig. 3. Receiver operating characteristic (ROC) curve analysis for prediction of mortality by serum B12 levels (‐‐‐ line), model for end-stage liver disease (MELD) scores (–– line) and their combination (⋯ line) at admission.
severity of the liver disease, patients with higher B12 concentrations had lower concentrations of serum albumin, and had higher INR, ALT and total bilirubin. Moreover, highly increased serum B12 concentration was associated with a higher rate of clinical complications such as cirrhosis and ascites (Table 1). We hypothesized that the association of B12 concentrations with mortality risk may, in part, be due to an effect of inflammation on liver and risk. A few limitations warrant consideration. Firstly, this was a singlecenter study and our sample size may have posed a limitation of this study. The findings need to be confirmed in multi-center and prospectively designed studies. Secondly, vitamin B12 was not dynamically observed, and thus, whether RDW values are stepwise increased when patient's condition is progressively deteriorated remains unclear. In conclusion, the study confirms a high prevalence of abnormally increased serum B12 concentrations in AoCLF patients. The determinants of increased serum B12 concentrations were in association with liver injuries. Our data identify a poor prognostic influence of increased serum B12 concentrations in AoCLF patients. The molecular marker of increased B12 concentrations adds predictive value to the conventional clinical factors such as MELD score, which in combination may be prognostically useful for early detection of disease progression and better treatment strategies.
Table 2 Cox proportional hazards analysis for predictors of death. Univariable
B12 (pg/ml) MELD score Age (y) Total protein (g/L) Albumin (g/L) ALT (U/L) Creatinine (mmol/L) Total bilirubin (μmol/L)
Multivariable
Odds ratio
95% CI
P
Odds ratio
95% CI
P
1.980 2.889 1.033 0.894 0.827 1.106 1.018 1.008
1.485–2.159 2.288–3.728 1.000–1.007 0.829–0.965 0.748–0.914 1.097–1.201 1.018–1.072 1.005–1.012
b0.001 b0.001 0.053 0.004 b0.001 0.013 0.001 b0.001
1.891 2.512 – – – – – –
1.329–2.049 1.977–3.153 – – – – – –
0.021 0.005 NS NS NS NS NS NS
Abbreviations: MELD score, model for end-stage liver disease score.
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