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Philip Hilgard,
Guido Gerken, Department of Gastroenterology and Hepatology,
University Hospital Essen, Essen, Germany
Correspondence to: Dr. Philip Hilgard, Universitatsklinikum
Essen, Klinikfur Gastroenterologie and Hepatologie, Hufelandstrabe
55, 45122 Essen, Germany. philip.hilgard@uni-essen.de
Telephone: +49-201-723-2390
Fax: +49-201-723-5971
Received: 2004-05-10
Accepted: 2004-08-12
Abstract
Nonspherocytic hereditary anemias are occasionally accompanied by
significant iron overload but the significance for the development
of chronic liver disease is not clear. We described two cases of
patients with chronic liver disease and severe iron overload due to
chronic hereditary hemolysis. Both patients have had signs of liver
cirrhosis and severe hemolysis since childhood. A hereditary
pyruvate kinase deficiency (PKD) was discovered as the underlying
reason for the hemolysis. Sequencing of the pyruvate kinase gene
showed a mutation within exon 11. Liver histology in both patients
revealed cirrhosis and a severe iron overload but primary
hemochromatosis was excluded by HFE-gene analysis. An iron reduction
therapy with desferrioxamine led to significant decrease of serum
ferritin and sustained clinical improvement. PKD-induced hemolysis
may cause severe iron overload even in the absence of HFE-genotype
abnormalities. This secondary iron overload can lead to chronic
liver disease and cirrhosis. Therefore, the iron metabolism of PKD
patients has to be closely monitored and iron overload should be
consequently treated.
ã 2005
The WJG Press and Elsevier Inc. All rights reserved.
Key words:
Hemochromatosis; Pyruvate kinase deficiency; Liver cirrhosis; Iron
overload; Desferrioxamine
Hilgard P, Gerken G. Liver cirrhosis as a consequence of iron
overload caused by hereditary nonspherocytic hemolytic anemia.
World J Gastroenterol 2005;
11(8): 1241-1244
http://www.wjgnet.com/1007-9327/11/1241.asp
INTRODUCTION
Hereditary hemolytic anemia is classified as spherocytic anemia and
nonspherocytic anemia. Spherocytic hemolytic anemia is based on an
impairment of the erythrocyte membrane or mutations of integral
compounds or the hemoglobin molecule. Nonspherocytic hemolytic
anemia is caused by a functional defect of particular enzymes of the
erythrocellular glucose metabolism, namely the glycolytic cascade.
Since, glycolysis is the only way for mature erythrocytes to
generate ATP, inhibition of this pathway results in disturbance of
the erythrocellular energy metabolism and, ultimately, hemolysis[1].
Here, we report two cases of patients with severe, non-spherocytic
hemolytic anemia caused by hereditary pyruvate kinase deficiency (PKD).
The chronic hemolysis led to secondary hepatocellular iron overload
and subsequently liver cirrhosis. These results suggest a consequent
control of the iron metabolism and the liver function in patients
with PK deficiency and, if necessary, an iron reducing therapy.
CASE REPORT
Case 1
A 56-year-old male patient (patient 1) with severe abdominal pain,
fever, nausea and emesis was admitted to the emergency room. In
addition, the patient had stigmata of chronic liver disease such as
icterus, spider naevi, dilated abdominal subcutaneous veins and
ascites. The clinical picture and an abdominal ultrasound
examination suggested advanced liver cirrhosis and acute
cholecystitis in the presence of gallstones. Laboratory parameters
at admission confirmed severe inflammation. A significant iron
overload, as indicated by high serum iron and ferritin and
siderinuria, was found. Bilirubin was high and the liver function
parameters (cholinesterase, prothrombin time and albumin) were
significantly altered. However, further differentiation of the
elevated bilirubin revealed that the major proportion was indirect
bilirubin (>70%). In conjunction with an elevated
lactat-dehydrogenase (LDH 450 U/L) this was suggestive for hemolysis.
Concomitantly, the patient had anemia (hemoglobin 9.4 mg/dL) with
significantly stimulated erythropoiesis, as demonstrated by the
elevated reticulocyte count of 21% (Table 1).
The patient reported the first onset of
icterus at the age of 12. At that time the spleen was removed and he
had significant and long-term improvement of the clinical situation.
Importantly, the patient revealed a positive family history with a
brother (patient 2) who suffered from chronic hemolysis as well.
Therefore, further efforts attempting to clarify the etiology of the
hemolytic anemia focused on the hereditary hemolytic syndromes.
Normal hemoglobin electrophoresis and negative testing for sickle
cell disease excluded hemoglobin abnormalities and no sphero or
elliptocytosis was found. Osmotic resistance of the red blood cells
was normal. However, examination of intracellular erythrocyte
enzymes revealed a massively decreased activity of pyruvate kinase
(3.5 U/1011
erythrocytes; normal: 17.1-26.5), while the activities of
glucose-6-phosphate dehydrogenase, hexokinase and glutation
reductase were normal. This finding led to the diagnosis of
hereditary PKD.
After the antibiotic pretreatment, open
cholecystectomy was performed. The gallbladder was severely inflamed
and the intraoperative inspection and palpation of the liver were
suggestive for liver cirrhosis. The liver was biopsied and histology
confirmed advanced periportal bridging. Furthermore, a severe iron
overload of hepatocytes and biliary epithelial cells was present,
suggestive for genetic hemochromatosis (Figure 1). However, analysis
of the HFE-genotype revealed no mutations and other chronic
liver diseases, such as alcohol, viral, or autoimmune-related
hepatitis and biliary diseases were excluded. Since a causal
treatment of PKD is at present not available, an iron-chelating
therapy with subcutaneous administration of desferrioxamine twice a
day[2]
was initiated. Despite a significant stabilization of the clinical
condition under therapy (no ascites was found in follow-up visits
after 3, 6 and 12 mo) and in view of the advanced stage of the liver
cirrhosis (Child-Pugh Score 9), the patient was evaluated and
recommended for liver transplantation. Unfortunately, he refused
transplantation and conservative therapy with desferrioxamine was
continued. Unexpectedly, liver function is to date stable for more
than 3 years.
Figure
1
Liver histology of two patients with hereditary corpuscular
hemolysis. Liver biopsies from patient 1 and 2 were fixed in
formaldehyde and embedded in paraffin following standard protocols.
Sections of 4-mm
thickness were stained either with hematoxylin-eosin
(HE-stain) or Perls Prussian Blue. Hepatic iron deposition was
graded according to DiBisceglie.
Case 2
The diagnosis of PKD prompted us to examine the younger brother of
the patient, who had a very similar medical history of the
development of icterus and concomitant splenectomy during childhood.
His actual laboratory parameters are shown in Table 1 (patient 2).
The bilirubin elevation in this patient was caused solely by
hemolysis, since direct bilirubin proportion was within the normal
range (0.7 mg/dL). Correspondingly, all other hemolysis parameters
were elevated and erythropoiesis was significantly stimulated (reticulocyte
count of 22). Pyruvate kinase activity was even lower in this
patient than in the older brother. The definitive confirmation of
the congenital defect in both patients was now performed by sequence
analysis of the pyruvate kinase gene within chromosome 5. A mutation
of a single base at position 1 529 within exon 11 of this gene, with
substitution of guanine (G) by adenosin (A) was evident (Figure 2).
On the amino acid level this mutation could cause an exchange of
arginine 510 into glutamate. This patient also had elevated liver
enzymes but, in contrast to the older brother, he had no clinical
signs of cirrhosis and his liver function was completely normal.
There was no evidence for alcoholtoxic, viral or autoimmune
hepatitis or an underlying biliary disease. However, iron metabolism
parameters with high serum iron and ferritin were in a similar range
as in his older brother, indicating massive iron overload. Liver
biopsy confirmed the severe hepatocellular iron overload, and
revealed furthermore advanced periportal bridging (Figure 1). This
patient showed a markedly reduced ejection fraction in
echocardiography, indicating iron-induced toxic cardiomyopathy. An
iron chelating therapy with desferrioxamine was initiated and
complemented by phlebotomy with a bimonthly withdrawal of 500-mL
blood. In both patients, the iron reducing therapy led to a
significant reduction of iron and ferritin over the next 12 mo
(Figure 3).
Figure 2(PDF)
Partial sequence analysis of the pyruvate kinase gene (exon
11) in the patients described. Genomic DNA was prepared from
peripheral blood mononuclear cells (PBMC) and sequenced according to
standard protocols, applying [a35S]
dATP during the termination reaction and subsequent fluorography.
Figure
3(PDF)
Decrease of iron overload in the patients under
desferrioxamine therapy. Iron metabolism was assessed by serum
ferritin concentration over a period of time of 30 mo.
Table 1
Laboratory parameters of the described patients at admission
| Liver
inflammation and
function |
Normal |
Patient
1 |
Patient
2 |
| ALT
(GOT), |
520
U/L |
30
U/L |
31
U/L |
| AST
(GPT) |
525
U/L |
39
U/L |
41
U/L |
| g-GT |
530
U/L |
41
U/L |
61
U/L |
| Alkaline
phosphatase |
<180
U/L |
224
U/L |
219
U/L |
| Prothrombin
time |
70-110% |
48% |
102% |
| INR |
|
1.43 |
0.99 |
| Albumin |
(3.65.0
g/dL) |
2.9
mg/dL |
4.4
mg/dL |
| Cholinesterase |
(3.57.0
kU/L) |
2.1
kU/L |
4.8
kU/L |
| C-reactive
protein |
<0.7
mg/dL |
10
mg/dL |
0.5
mg/dL |
| Erythrocyte
morphology |
|
anisocytosis, |
macrocytosis, |
| |
|
poikilocytosis, |
anisocytosis, |
| |
|
acanthocytosis |
poikiLocytosis |
| Blood
count, parameters of hemolysis and iron metabolism |
| WBC |
(310/nL) |
24
/nL |
13
/nL |
| Hemoglobin |
(13.5-16
g/dL) |
9.2
g/dL |
11.5
g/dL |
| Free
hemoglobin |
(35-65
mg/dL) |
150
mg/L |
115
mg/L |
| Total
bilirubin |
(0.4-1.0
mg/dL) |
8.3
mg/dL |
7.6
mg/dL |
| Direct
bilirubin |
(0.2-0.5
mg/dL) |
1.7
mg/dL |
0.8
mg/dL |
| Haptoglobin |
(<0.23
g/L) |
<
0.233 g/L |
<
0.233 g/L |
| Lactat-dehydrogenase |
(10-150
U/L) |
450
U/L |
375
U/L |
| Reticulocytes |
(<1%) |
21% |
22% |
| Ferritin |
(15630
ng/mL) |
1
850 ng/mL |
1
620 ng/mL |
| Urine
iron excretion |
|
0.5
mg/24 h |
0.3
mg/24 h |
DISCUSSION
The diagnosis of PKD in both cases was confirmed by sequence
analysis of the PK gene, which revealed a single nucleotide mutation
(G to A) in the PK gene within exon 11 at position 1 529. Together
with the glucose-6-phosphate-dehydrogenase (G6PD) deficiency, PK
deficiency is the most common type of hereditary enzyme defect
within the glycolytic pathway causing nonspherocytic anemia[3,4].
The point mutation detected in our 2 patients (G1529A) is one of the
most frequent mutations within the western European population[5-7].
The remaining activity of PK in patients carrying this mutation has
been reported to be 10-25% and reticulocytosis varies from 5% to 66%[7].
The disease shows a broad range of clinical severity, which is
loosely correlated to the impairment of hematological parameters:
The lower the residual PK activity and the higher the reticulocyte
count, the more the patients are affected[7,3].
Since PK catalyzes the final step within the glycolytic pathway (phosphoenol-pyruvate→pyruvate),
a decreased activity results in alterations of the erythrocellular
energy metabolism. Subsequently, the activity of the membrane
associated sodium-potasium ATPase is impaired, leading to
instability of red blood cells[8].
A main clinical feature in the two PKD
patients, examined in this study, is a considerable iron overload.
The iron overload is independent of transfusion therapy. In the
liver tissue, the iron overload includes hepatocytes and even
biliary epithelial cells. Since this histological pattern correlates
to genetic hemochromatosis, the HLA phenotype and the commonly known
mutations in the hereditary hemochromatosis (HFE)-gene (C282Y
and H63D) were examined. The possibility of a coincidence of both
diseases seems not unlikely, since 2-20% of the European population
have mild alterations of the iron status associated with
heterozygosity for the main hemochromatosis mutation (C282Y), while
a much higher proportion carry the H63D mutation. A recent study
revealed abnormalities of the HFE-gene in 35% of
nontransfused patients with PKD and these abnormalities coincide
with increased serum iron and ferritin[9].
However, in our patients heterozygosity for genetic hemochromatosis
was ruled out by a normal HFE-genotype.
In addition to heterozygosity for
hemochromatosis, splenectomy has been reported to be an independent
risk factor for iron overload in hemolytic diseases[10,11].
While the increased iron turnover is the basis for the iron overload
in PKD[9,12],
it alone is not sufficient to cause iron accumulation in this
disease[9,12].
Since both of our patients underwent splenectomy during childhood,
this circumstance was considered as a main contribution to the
significant iron overload they had. It is well accepted, that
primary and secondary iron overloads have prefibrogenic effects
within the liver tissue, by sustained induction of oxidative stress[13,14].
As in our patients, the level of the liver transaminases may only be
marginally elevated, since the iron-induced chronic inflammation is
often of very low activity, but nevertheless results in progressive
fibrosis[13].
Independent of the pathogenesis, liver iron concentration must
exceed a critical threshold, which has been suggested to be around
350-400 mmol/L/g,
in order to cause hepatocellular damage[15,16].
Above this threshold, the secondary iron overload (e.g. due to
hemolytic disease) may have the same deleterious effects as the
primary iron overload in genetic hemochromatosis.
Taken together, it becomes obvious that the clinical symptoms of the
patients described in this study, are all based on the PKD in
association with the splenectomy. Under this circumstance, chronic
hemolysis is capable of causing significant iron overload, which in
turn is responsible for the liver and cardiac disease of both
patients. The pathophysiological importance of iron overload in
these patients was also demonstrated by the effectiveness of the
iron chelating therapy, which led to a significant decrease of serum
ferritin and a stable liver function for more than 3 years. In
addition to the iron overload, PKD-induced hemolysis is responsible
for the abdominal symptoms of the elder patient (case 1). The
chronic excess of bilirubin obviously altered the solvent properties
of the bile, resulting in cholelithiasis, presumably consisting of
bilirubin stones. The stones caused cholecystitis, perhaps combined
with acute or chronic cholangitis. After cholecystectomy, these
associated symptoms such as fever and abdominal pain relieved
completely. Histological examination of gallbladder and the
gallstones after surgery confirmed these clinical suspicions.
In
summary, this study demonstrates that PKD-induced hemolysis in
conjunction with splenectomy may cause severe iron overload even in
the absence of HFE-genotype abnormalities. The secondary iron
overload may lead to chronic liver disease and cirrhosis.
Unfortunately, a sufficient causal therapy for PKD is at present not
available, although new therapeutic strategies are under evaluation.
The most promising gene therapeutic approaches may be either
transplantation of heterologous hematopoietic stem cells[17]
or the reinfusion of autologous stem cells after correction of the
PK gene sequence in the stem cell DNA in vitro[18].
Until such therapies become available, we suggest a close monitoring
of the iron metabolism of PKD patients and a consequent treatment of
iron overload.
ACKNOWLEDGEMENTS
The authors thank Peter Nuernberg, Institute of Medical Genetics,
Charit Universitiy Hospital, Humboldt University, Berlin, Germany
for sequencing the pyruvate kinase gene and Peter Schirmacher,
Institute for Pathology, University of Cologne for staining of the
liver tissue.
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