Original Article

Yonago Acta Medica 2017;60:31–39

Falsely Elevated Serum Vitamin B12 Levels Were Associated with the Severity andPrognosis of Chronic Viral Liver Disease
Takaaki Sugihara, Masahiko Koda, Toshiaki Okamoto, Kenichi Miyoshi, Tomomitsu Matono, Kenji Oyama,
Keiko Hosho, Jun-ichi Okano, Hajime Isomoto and Yoshikazu Murawaki

Division of Medicine and Clinical Science, Department of Multidisciplinary Internal Medicine, School of Medicine, Tottori University
Faculty of Medicine, Yonago 683-8504, Japan

ABSTRACT

Vitamin B12 is stored primarily in the liver; this vitaminis essential for one-carbon metabolism and cell division. It acts as a cofactor for two enzymatic reactions,
namely, methionine synthesis from homocysteine andsuccinyl-CoA synthesis from methylmalonyl-CoA, inmammalian systems.1 Some studies have indicated thatelevated serum levels of vitamin B12 might be a sign ofa serious and life-threatening disease. Such falsely highvalued of serum vitamin B12 levels are observed in myeloproliferative disease, acute hepatitis, severe alcoholic
liver disease, and cirrhosis.2–6
Vitamin B12 binds with transcobalamin (TC) II. The
complex holoTC II, which is the biologically active formof vitamin B12, is recognized by specific receptors on
all cell types. The active form of vitamin B12 comprises
only 6–20% of the total serum vitamin B12 level. The remaining major portion (70–90%) is bound to haptocorrin (HC); this is named holohaptocorrin (holoHC) andis the inactive form of vitamin B12, which is stored in
the liver.7, 8 Several reports have reported that the falsely
high values are due to increasing the holoHC levels.3
In our experience, we have observed highly elevatedvitamin B12 levels in patients with advanced stages of viral liver cirrhosis. There are only one report that demonstrated the association of vitamin B12 level with disease
severity and mortality in hepatitis B virus infection withshort term.9 No reports have evaluated the dynamics ofvitamin B12 in chronic viral hepatitis. Accordingly, thepresent study aimed to evaluate the relationship of serum
vitamin B12 between the disease severity and long term
prognosis of liver disease in patients with viral hepatitis
and cirrhosis.

Background

Vitamin B12 is stored primarily in theliver, and highly elevated serum vitamin B12 levels occur
in acute hepatitis and severe alcoholic liver disease. We
evaluated the relationship between vitamin B12 levels
and liver disease severity and long term prognosis in patients with chronic viral hepatitis and cirrhosis.
Methods We enrolled 90 patients (57 men, 33 women)with chronic viral hepatitis and cirrhosis who admitted
to our hospital as a prospective cohort study. Overall,
37 patients had chronic hepatitis and 53 had cirrhosis(Child-Pugh A 33, B 13, and C 7); 57 patients had primary liver cancer. Serum vitamin B12 concentration andholotranscobalamin (holoTC) II (active form of vitaminB12) were determined and followed prospectively for atleast 5 years.
Results Mean total serum vitamin B12 concentration
was significantly higher in Child-Pugh C (1308 ± 599
pg/mL) compared to those with chronic hepatitis (655
± 551 pg/mL), Child-Pugh A (784 ± 559 pg/mL), andChild-Pugh B (660 ± 464 pg/mL) (P = 0.036) Presenceof primary liver cancer also influenced serum vitamin
B12 levels [657 (167–2956) vs. 432 (189–2956); P = 0.015].
Patients were divided into quartiles by vitamin B12 level.
Patients without primary liver cancer in quartile 4 (≥880 pg/mL) demonstrated significantly poorer prognosis
than those in quartiles 1–3 (< 880 pg/mL) (P = 0.023).
The percentage of holohaptocorrin (holoHC) [(total vitamin B12 – holoTC II) × 100] was significantly higher inChild-Pugh B and C 86 (80–87)% than chronic hepatitis
and Child-Pugh A 77 (31–89)% (P = 0.006) Multivariate
analysis indicated serum vitamin B12 levels (HR = 1.001,
P = 0.029) as a prognostic factor.
Conclusion Falsely elevated serum vitamin B12 levels
mainly composed of increased holoHC were associatedwith severity (Child-Pugh C and primary liver cancer)
and prognosis in chronic viral liver disease.

Corresponding author: Takaaki Sugihara, MD, PhD
sugitaka@med.tottori-u.ac.jp
Received 2016 December 8
Accepted 2017 January 4
Abbreviations: AFP, alpha-fetoprotein; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CD, cluster of differentiation;
DCP, des-γ-carboxy prothrombin; HBV, hepatitis B virus; HC,
haptocorrin; HCC, hepatocellular carcinoma; HCV, hepatitis C
virus; holoHC, holohaptocorrin; holoTC, holotranscobalamin; HR,hazard ratio; NFκB, nuclear factor kappa B; NK, natural killer; TC,transcobalamin; TNF-α, tumor necrosis factor-alpha; TNM, TNM
classification of malignant tumours

Key words

holotranscobalamin; liver cirrhosis; prognosis; viral hepatitis; vitamin B12

31

T. Sugihara et al.

SUBJECTS AND METHODS
Subjects

Cox proportional hazards model. Statistical significancewas set at P < 0.05.

From March 2007 to March 2010, consecutive patientswith chronic viral hepatitis and viral induced cirrhosis
were prospectively enrolled in the present study. Patients
who underwent a total gastrectomy, vegetarians andthose receiving nutrient supplementation, including vitamin B12, were excluded from the study.

RESULTS
Patient background

Ninety patients [57 men, 33 women; median age, 69(range, 30–88) years] were enrolled this study. Of thesepatients, 60 (67%) had hepatitis C virus (HCV) and 30
patients (33%) had hepatitis B virus (HBV). Twenty
patients (22%) habitually consumed ≥ 20 g/day alcohol.
Seven patients (8%) had heart disease (e.g. ischemic
heart disease, valvular heart disease, and arrhythmia).
Nine patients (10%) underwent distal gastrectomy. Underlying liver disease was chronic hepatitis in 37 patients(41%) and cirrhosis in 53 (59%) (Table 1).

Methods

Cirrhosis was diagnosed according to clinical, biochemical, and/or imaging findings. The severity of cirrhosis
was categorized using the Child-Pugh classification.10
The presence of primary liver cancer was also recorded.
The stages of hepatocellular carcinoma (HCC) were categorized by TNM staging. Blood samples were collected
at the date of inclusion, and hemoglobin, blood chemistry [albumin, total bilirubin, aspartate aminotransferase(AST), alanine aminotransferase (ALT)], prothrombin
activity percentage, alpha-fetoprotein (AFP), and des-γcarboxy prothrombin (DCP), and serum vitamin B12 levels were determined. Total serum vitamin B12 concentration was determined by chemiluminescent immunoassay(CLIA: Access Vitamin B12; beckman coulter, Tokyo,
Japan). Levels of holoTC II were also measured by microparticle enzyme immunoassay (AxSYM Active-B12;
abbott diagnostics, Abbott Park, IL). The percentage of
holoTC II was expressed as the ratio of holoTC II to total
serum vitamin B12. The percentage of holoHC was calculated as (total vitamin B12 – holoTC II) × 100. All thepatients were followed prospectively for at least 5 years
and evaluated for prognosis.

Table 1. Clinical findings in study subjects
Patients

n = 90Sex (Male:Female)
57:33
Age (years)
69 (30–88)
Etiology
30 (33%)
HBV infection
HCV infection
60 (67%)
Alcohol consumption (≥ 20 g/day)
20 (22%)Comorbidity
7 (8%)
Heart disease
Gastrectomy (distal)
9 (10%)Atrophic gastritis
33 (37%)The severity of underlying liver disease37 (41%)Chronic hepatitis
Cirrhosis
53 (59%)Child-Pugh classification
33:13:7(A:B:C)Primary Liver Cancer (HCC:ICC)
56:1 (62%)
TNM Stage
18:11:17:4:5:2( I:II:II:IVA:IVB:unknown)
Laboratory values(g/dL)
12.3 (6.5–16.9)
Hemoglobin
MCV(fL)
95.8 (62.4–112.9)
MCHC(%)
34.4 (31.4–36)
Albumin(g/dL)
3.6 (2.1–5.1)
AST(IU/L)
47 (14–847)
ALT(IU/L)
37 (7–1073)
Total bililubin(mg/mL)
0.8 (0.3–15.1)
Prothrombin percent activity (%)
86.9 (5–117.1)
Total serum vitamin B12(pg/mL) 584.5 (160–2956)
AFP(ng/mL)
8.7 (1.2–501570)
DCP(mAU/mL) 24 (10–84980)
Data are expressed as median (range).
AST, aspartate aminotransferase; ALT, alanine aminotransferase;
AFP, alpha fetoprotein; DCP, Des-gamma-carboxy prothrombin;
HBV, hepatitis B virus; HCV, hepatitis C virus; HCC, hepatocellular carcinoma; ICC, intrahepatic cholangiocellular carcinoma;
MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration; TNM, TNM classification of malignant
tumours.

Ethical Considerations

The study protocol confirmed to the ethical guidelinesof the Helsinki Declaration of 1975, as revised in 2000,
and was approved by the ethics committee of the Tottori
University (No. 1231). Patients were enrolled after giving their written informed consent.
Statistical analysis

Data are expressed as median (range) or mean ± SD.
Statistical analyses for significant differences among
the groups were performed using the chi-square test,
Student’s t test, Mann-Whitney’s U test, ANOVA, andKruskal-Wallis Test. F test and Bartlett’s test was used
to assess the equality of variances. Correlations were
calculated using Pearson’s product-moment correlation
coefficient. Cumulative survival rate was calculated by
the Kaplan-Meier method, and significant differences
between two groups were calculated using the Log rank
test. Multivariate analyses were performed using the32

Falsely elevated vitamin B12 in viral liver disease

Vitamin B12 analysis(pg/mL)
2500

Serum vitamin B12 level

Serum vitamin B12 levels were significantly higher
in patients with cirrhosis than chronic hepatitis [647(160–2956) vs. 461 (189–2956) pg/mL (P = 0.029)], andit was particularly in patients categorized as Child-Pugh
C (1308 ± 599 pg/mL) than those with chronic hepatitis(655 ± 551pg/mL), Child-Pugh A (784 ± 559 pg/mL),
and Child-Pugh B (660 ± 464 pg/mL) (P = 0.036) (Fig.1).
The vitamin B12 levels were also higher in patients withprimary liver cancer than those without [657 (167–2956)
vs. 432 (189–2956) pg/mL, P = 0.015] (Table 2).
Then we divided the patients with and without primary liver cancer. Among the patients with primaryliver cancer, the vitamin B12 levels were found to besignificantly higher in drinkers (alcohol consumption≥ 20 g/day) than non-drinkers [885 (160–2956) vs. 625(263–2265) pg/mL, P = 0.025]. On the other hand,among the patients without primary liver cancer, it wassignificantly higher in patients with hepatitis B virus infection than hepatitis C virus infection [655 (272–2956)
vs. 392 (189–1083) pg/mL, P = 0.018], and the patientswith cirrhosis than chronic hepatitis [655 (379–2956) vs.
386 (189–1200) pg/mL, P = 0.004] (Table 3).
Moreover, we also divided the patients with chronic
hepatitis and cirrhosis to separate the influence of liverfunction on vitamin B12 for each parameter. Vitamin B12levels were significantly higher in primary liver cancer
only in patients with chronic hepatitis [731 (196–2956)
vs. 386 (189–1200) pg/mL, P = 0.009] (Table 4).
To evaluate the relationship of serum vitamin B12
with the patients’ clinical and biochemical parameters,
the correlations between serum vitamin B12 levels andthose parameters (age, hemoglobin, mean corpuscularvolume, mean corpuscular hemoglobin concentration,
albumin, AST, ALT, total bilirubin, prothrombin activ-

*

2000

*

*

1500

1000

500

0

CH(37) A(33)

B(13) C(7)Child-PughFig. 1. Serum vitamin B12 levels in patients with chronic viral
hepatitis. Serum vitamin B12 levels were significantly higher inpatients categorized as Child-Pugh C (1308 ± 599 pg/mL) thanthose in CH (655 ± 551 pg/mL), Child-Pugh A (784 ± 559 pg/mL),
and Child-Pugh B (660 ± 464 pg/mL) (P = 0.036). *P < 0.05. CH,
chronic hepatitis.

ity percentage, AFP, and DCP) were estimated using
Pearson’s product-moment correlation coefficient. Serum
vitamin B12 levels were positively correlated with AFP (r= 0.231, P = 0.04), negatively prothrombin activity percentage (r = –0.248, P = 0.017) (Table 5).

Table 2. Influence of clinical factors on serum vitamin B12 levels
Serum vitamin B12 (pg/mL)
All patients (n = 90)

P value

Sex(Male:Female)581 (196–2956):640 (160–2956)
n.s.
Etiology(HCV:HBV)
513 (189–2956):656 (160–2956)
n.s.
Alcohol ≥ 20g/day(Yes:No)
660 (160–2956):520 (263–2265)
n.s.
Heart disease(Yes:No)431 (320–1298):410 (160–2956)
n.s.
Gastrectomy*(Yes:No)
607 (160–2956):367 (263–1494)
n.s.
Cirrhosis(Yes:No)
647 (160–2956):461 (189–2956)
0.029
Primary liver cancer(Yes:No)657 (160–2956):432 (189–2956)
0.015
TNM Stage(I/II:III/IVA/IVB)
549 (233–2956):728 (160–2265)
n.s.
Data are expressed as median (range). *Distal gastrectomy.
HBV, hepatitis B virus; HCV, hepatitis C virus; TNM, TNM classification of malignant tumours; n.s., not significant.

33

T. Sugihara et al.
Table 3. Influence of clinical factors on serum vitamin B12 levels between patients with and without primary livercancerSerum vitamin B12 (pg/mL)
Patients with primary liver cancer(n = 57)

P value

Patients without
primary liver cancer (n = 33)

Sex
632 (196–2956):686 (160–1957)
n.s.
461 (277–1200):410 (189–2956)(Male:Female)
Etiology
664 (205–2956):657 (160–2029)
n.s.
392 (189–1083):655 (272–2956)(HCV:HBV)
Alcohol ≥ 20 g/day
885 (160–2956):625 (263–2265)
0.025
432 (299–1083):410 (189–2956)(Yes:No)Heart disease819 (160–2956):647 (320–1298)
n.s.
581 (581–581):424 (189–2956)(Yes:No)
Gastrectomy*
320 (263–1494):686 (160–2956)
n.s.
283 (367–379):461 (189–2956)(Yes:No)
Cirrhosis
645 (160–2265):731 (196–2956)
n.s.
655 (379–2956):386 (189–1200)(Yes:No)TNM Stage
549 (233–2956):728 (160–2265)n.s.
—( I/II:III/IVA/IVB)
Data are expressed as median (range). *Distal gastrectomy.
HBV, hepatitis B virus; HCV, hepatitis C virus; TNM, TNM classification of malignant tumours; n.s., not significant.

P value
n.s.
0.018
n.s.
n.s.
n.s.
0.004
—

Table 4. Influence of clinical factors on serum vitamin B12 levels between patients with chronic hepatitis andcirrhosis
Serum vitamin B12 (pg/mL)
Patients with chronic hepatitis (n = 37)

P value

Patients with cirrhosis (n = 53)

Sex509 (196–2956):189 (189–1298)
n.s.
631 (205–2265):647 (160–2956)(Male:Female)
Etiology
432 (189–2956):634 (196–1298)
n.s.
640 (205–2265):656 (160–2956)(HCV:HBV)
Alcohol ≥ 20 g/day
461 (299–1298):449 (189–2956)
n.s.
1083 (263–2265):624 (160–2956)(Yes:No)Heart disease
634 (320–1298):432 (189–2956)n.s.
952 (367–1292):645 (160–2956)(Yes:No)
Gastrectomy*
344 (320–367):483 (189–2956)
n.s.
263 (160–2956):379 (263–1494)(Yes:No)Primary liver cancer
731 (196–2956):386 (189–1200)
0.009
645 (160–2265):655 (379–2956)(Yes:No)TNM Stage
526 (233–2956):731 (196–1417)
n.s.
751 (263–1957):472 (160–2965)( I/II:III/IVA/IVB)
Data are expressed as median (range). *Distal gastrectomy.
HBV, hepatitis B virus; HCV, hepatitis C virus; TNM, TNM classification of malignant tumours; n.s., not significant.

HoloTC II analysis

P value
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.

significantly higher in Child-Pugh B and C 86 (80–87)%
compared with chronic hepatitis and Child-Pugh A 77
(31–89)% (P = 0.006) (Fig. 2B).

In 43 of 90 patients, levels of holoTC II were evaluated.
We found that the percentage of holoTC II tended todecrease with progression of liver disease. The medianpercentages of holoTC II were 22(11–69)% in patientswith chronic hepatitis (n = 25), 24 (19–37) % in ChildPugh A (n = 13), 16 (13–20)% in Child-Pugh B (n =4), and 13% in Child-Pugh C (n = 1), and the statisticalsignificance was indicated between Child-Pugh A and
B (P = 0.031) (Fig. 2A). The percentage of holoHC was

Survival analysis

During the observation period (median, 54.3 months;
range, 0.6–101.7months), the overall survival rates were89% at 1 year, 81% at 2 years, and 67% at 5 years. Cirrhotic patients had significantly poorer prognosis compared with non-cirrhotic patients [33.5 (0.6–101.7) vs.
34

Falsely elevated vitamin B12 in viral liver disease

66.9 (3.1–72.1) months, P = 0.003]. Patients with primary liver cancer had also significantly poorer prognosis
compared to those without them [28.5 (0.6–101.7) vs.
67 (15.6–71.7) months, P < 0.001]. Ninety patients weredivided into quartiles Q1 to Q4, according to serum
vitamin B12 level: Q1, < 403.25; Q2, 403.25–582; Q3,
583–879; and Q4, ≥ 880 pg/mL (Fig. 3A). Among all
the patients, Q4 had markedly low cumulative survival

rate than those Q 1-3 (P = 0.058) (Fig. 3B). On the other
hand, among the patients without primary liver cancer,
Q4 had significantly low cumulative survival rate than
Q 1-3 (P = 0.003) (Fig. 3C). Univariate analysis revealed
that serum albumin, total bilirubin, AFP > 10 ng/mL,
DCP > 40 AU/mL, and serum vitamin B12 level weresignificant prognostic factors for the overall survival
rate. The stepwise multivariate Cox proportional hazards
model showed that the independent factors contributing
to cumulative survival rate were serum albumin, DCP >40 AU/mL and serum vitamin B12 levels (Table 6).

Table 5. Correlations between serum vitamin B12levels and clinical/biochemical parametersr

Patients (n = 90)

P valueDISCUSSION
Our study demonstrated two important findings. First,
serum vitamin B12 levels were significantly elevated inpatients categorized as Child-Pugh C and with primary liver cancer. Second, serum vitamin B12 level was asignificant independent predictor for overall survival inpatients with chronic viral liver disease.
We found that serum vitamin B12 levels were significantly higher in cirrhotic patients with Child-Pugh C
than in patients with chronic hepatitis or Child-Pugh A/
B cirrhosis. It has been previously reported that highly
elevated vitamin B12 levels in plasma were indicated inacute hepatitis, severe alcoholic liver disease, and cirrhosis.2–6, 11, 12 Our data indicate that serum vitamin B12 levels were falsely elevated with the severity also in chronicviral liver disease. Total serum vitamin B12 levels wereSex
–0.038n.s.
Age (year)–0.005n.s.
Hemoglobin (g/dL)–0.053n.s
MCV (fL)–0.018n.sMCHC (%)
0.001
n.sAlbumin (g/dL)–0.203n.s.
AST (IU/L)
0.168
n.s.
ALT (IU/L)
0.114
n.s.
Total bilirubin (mg/mL)
0.113
n.s.
Prothrombin percent activity–0.248
0.017
AFP
0.231
0.040
DCP
0.084
n.s.
AFP, alpha-fetoprotein; AST, aspartate aminotransferase; ALT,
alanine aminotransferase; BTR, branched-chain amino acids totyrosine ratio; DCP, des-γ-carboxy prothrombin; MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration; n.s., not significant.

(%)
100

80

80

60

Percentage of holoHC

B
(%)
100

Percentage of holoTC II

A

*

40

60

40

20

20

0

*

0
CH(25) A(13)

B(4)

C(1)

CH/ChildA(38)

ChildB/C(5)

Child-PughFig. 2. Percentage of holoTC II and holoHC.
A: Percentage of holoTC II was significantly different between Chid-Pugh A and B (P = 0.031). *P < 0.05. B: The percentage of holoHC[(total vitamin B12 – holoTC II) × 100] was significantly higher in Child-Pugh B and C 86 (80–87)% than chronic hepatitis (CH) andChild-Pugh A 77 (31–89)% (P = 0.006). **P < 0.01. holoHC, holohaptocorrin; holoTC, holotranscobalamin.

35

T. Sugihara et al.

Cumulative survival rate

Cumulative survival rate

A

Time (months)

Cumulative survival rate

Time (months)

Fig. 3. Cumulative survival rate calculated by the Kaplan-Meiermethod, by serum vitamin B12 level quartiles.
A: The 90 patients were divided into quartiles by vitamin B12level: < 403.25 (Q1, solid line); 403.25–582 (Q2, dotted line), 583–
879 (Q3, dashed line), and ≥ 880 pg/mL (Q4, dash-dotted line).
B: Cumulative survival rate differed between Q 1-3 (solid line)
and Q4 (dotted line) in all the patients (P = 0.058). C: Cumulative
survival rate differed significantly between Q 1-3 (solid line) andQ 4 (dotted line) in the patients without primary liver cancer (P =0.003).

Time (months)
Table 6. Univariate and multivariate analyses of factors predicting the overall survival rate
Factors
Sex
Age
Albumin
Total bilirubin
PT
AFP
DCP
Serum vitamin B12

year
g/dL
mg/dL
%
> 10 ng/mL> 40 AU/mL
pg/mL

Univariate
P value

Multivariate analysis
HR
95% CI

P value

0.374
0.369
< 0.001
0.006
0.086
0.003
< 0.001
0.034

0.368
1.231
1.010
1.546
2.682
1.001

0.008
0.170
0.457
0.340
0.033
0.029

0.176–0.767
0.915–1.658
0.984–1.036
0.632–3.781
1.084–6.636
1.000–1.001

CH,chronic hepatitis; AFP, alpha fetoprotein; CI, confidence interval; DCP, des-γ-carboxy prothrombin; HR, hazard
ratio; PT, prothrombin percent activity.

36

Falsely elevated vitamin B12 in viral liver disease

Normal	
  Liver 	

Adjust	  the	
  balance	
  of	
  holoTC	
  II	
  in	
  circula2on	

Circula2on	
Excess	  release?	

×	
Cirrhosis	

Reduced	  clearance?	

×	
Liver	
  cancer	

Fig.4
Fig. 4. Scheme of the hypothesis for falsely elevated vitamin B12 in liver disease.
HoloHC (■), the inactive form of vitamin B12, is stored in the liver. The stored holoHC adjusts holoTC II(▲), the inactive form of vitamin
B12, in circulation. There are possible two mechanisms responsible for falsely elevated serum vitamin B12 levels in liver diseases. First,
holoHC leaks into the circulation due to the destruction of hepatocytes (excess release hypothesis). Second, the reduced uptake of holoHC
by the injured liver (reduced clearance hypothesis). holoHC, holohaptocorrin; holoTC, holotranscobalamin.

significantly higher in cirrhotic patients. The biomarkerof hepatic reserve, such as prothrombin activity percentage, was found to be weakly correlated with the total
serum vitamin B12 levels in the present study.
Among the patients with primary liver cancer, thevitamin B12 level was higher in alcohol consumption ≥20 g/day group than in those were not. Alcoholic liverdisease have already shown to have the falsely highvitamin B12 levels associated with liver dysfunction.4, 5
On the other hand, among the patients without primaryliver cancer, serum vitamin B12 levels were significantly
higher in HBV than those in HCV patients. The reason
was the number of patients classified into Child-Pugh B
and C was significantly higher in HBV patients without
primary liver cancer (data not shown).
Total serum vitamin B12 levels were also significantly higher in patients with primary liver cancer thanthose without. Some reports have also indicated that
serum vitamin B12 levels are elevated in patients with
HCC.13–15 However, primary liver cancer is usually complicated with viral cirrhosis, the elevation should be distinguished from the association with cirrhosis. In present
study, the difference was demonstrated only in patientswith chronic hepatitis. Therefore, it could be concluded
that primary liver cancer directly associated with serum
vitamin B12 levels.

According to these findings, we could demonstratethat severe liver dysfunction (Child-Pugh C) and primary liver cancer affected the falsely elevated vitamin B12levels.
In this study, we analyzed holoTC II additionally insome patients to clarify the mechanisms of falsely elevation. Unfortunately, we could evaluate only one patientin Child-Pugh C. We observed significantly lower level(within normal range) of the holoTC II percentage in patients of Child-Pugh B and C. It also means the percentage of holoHC increased in Child-Pugh B and C.
Thus, we propose two mechanisms responsible forelevated serum vitamin B12 levels in these patients (Fig.
4). First, holoHC possibly leaks into the circulation due
to the destruction of hepatocytes (excess release hypothesis). This is supported by the observation that plasma
vitamin B12 levels are elevated in 25–40% cases of acutehepatitis.2, 11, 16, 17 However, in our study, serum vitamin
B12 levels did not correlate with transaminase levels, thishypothesis could not explain high serum vitamin B12 inchronic viral hepatitis. Alternatively, the reduced uptakeof holoHC by the injured liver may contribute to elevatedlevels of serum vitamin B12 (reduced clearance hypothesis). The asialoglycoprotein receptors, which bind holoHC for uptake into the liver, are expressed on normal
hepatocytes. Since the number of receptors decreases
37

T. Sugihara et al.

with the destruction of hepatocytes or the progression ofliver cancer, holoHC remains in the circulation.14, 15 The
study using liver biopsy specimens have shown that theloss of vitamin B12 storage in the liver is associated withincreased serum vitamin B12 levels.16, 18–21
In our study, elevated levels of serum vitamin B12
were also associated with poor prognosis. Some reports
have already indicated that vitamin B12 levels are associated with increased mortality in critically ill patientsof other diseases.22, 23 The mechanisms that affect the
prognosis of such patients remain unknown; however,based on the known aspects of hepatic inflammation andoxidative stress, it is possible to speculate on the mechanism. Previous reports have also shown that vitamin
B12 modulates inflammation. The relationship between
vitamin B12 and cytokine levels was demonstrated in humans.24 In the report, vitamin B12 deficiency was related
to high levels of tumor necrosis factor-alpha (TNF-α),and vitamin B12 supplementation normalized these levels. TNF-α is a well-known cytokine that plays a crucialrole in chronic inflammation as well as in chronic hepatitis.25, 26 Another report has indicated that vitamin B12
may regulate nuclear factor kappa B (NFκB), which istranscription regulator activated by cytokines like TNF-α,
and it determines cell survival and apoptosis.27, 28 These
reports suggest that vitamin B12 plays the role of a modulator for cytokine expression in the injured liver wherethe vitamin B12 storage has diminished.
Tamura et al. also indicated the role of vitamin B12
as an immunomodulator.29 demonstrating that vitamin
B12 administration increased the percentages of clusterof differentiation (CD) 8+ cells and natural killer (NK)cells in vitamin B12-deficient patients. Moreover, Birch etal. reported thiolatocobalamin (a vitamin B12 derivative)
has a protective effect on oxidant-damaged cells.30 Furthermore, the hepatoprotective effect of vitamin B12 hasbeen demonstrated using dimethylnitrosamine-induced
liver injury in a mouse model.31 This hepatoprotective
effect of vitamin B12 may be achieved by the maintenance of sulfhydryl levels under oxidative conditions.
Taken all things together, the association of falsely
elevated vitamin B12 with prognosis is considered to be
due to diminished hepatic storage (excess release or reduced clearance) and loss of its hepatoprotective effect.
However, it remains unclear whether the hepatic
vitamin B12 storage has any effect on maintain hepaticfunction in normal liver. The efficacy of vitamin B12
supplementation for cirrhosis also remains unknown.
Further studies are needed to clarify the role of vitamin
B12 in hepatic diseases.
A limitation of our study is the small number of patients. The prognosis of Q4 (vitamin B12 ≥ 880 pg/mL)

was significantly poor only in the patients without primary liver cancer; however, primary liver cancer is usually complicated with cirrhosis. To predict the prognosisof viral liver disease, appropriate cut-off level should bedetermined in all the patients. Large-scale study should
be conducted to clarify the cut-off level of serum vitamin B12 for viral liver disease.
In conclusion, falsely elevated serum vitamin B12levels mainly composed of its increased holoHC wereassociated with severity and prognosis in viral liver disease.
Acknowledgments: We would like to thank Younghee Koh of Abbott Japan Co. Ltd. for technical support and Yuki Fujiwara of thelaboratory department in Tottori University Hospital for technicalassistance.
The authors declare no conflict of interest.

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