S.A .
772
MEDICAL
J OU R NAL
10 Jun e 1972
(Supplemen t-Solllh African Journal of Laborato ry and Clinical M edicin e)
LCM 58
The Redox State of the Liv'er in
Symptomatic Porphyria *
B. C. SHANLEY
AND
S. M. JOUBERT, Department of Chemical Pathology, Uni versity of Natal, Durban
SUMMARY
EXPERIMENTAL APPROACH
Impaired galactose tolerance was found in 10 out of 11
adult Bantu patients with symptomatic porphyria. No
significant correlation was found between galactose TYz
and urinary porphyrin excretion or hepatic ALA synthetase
activity. These findings do not support the hypothesis
that there is a causal relationship between an altered
redox state of the liver cell and porphyrinogenesis in
symptomatic porphyria. It is concluded, however, that in
this disease galactose TV2 does not accurately reflect the
cytoplasmic NAD/NADH 2 ratio of the liver cell as is the
case in normal subjects .
Measurement of the total NAD and NADH2 content of
whole liver by direct means is unsatisfactory, since this
gives no information on the relative amounts of free and
bound nucleotides or on their intracellular distribution ,
which is heterogeneous' Methods employing indirect measurements are far more useful, such as the determination
of the lactate/pyruvate ratio. These two metabolites represent the reduced and oxidized substrates respectively of a
cytoplasmic NAD-linked dehydrogenase system and they
are in equilibrium with the nucleotides.' However, this
type of measurement also was obviously not readily applicable to a patient study.
The most convenient approach was the use of the
galactose tolerance test as recommended by Forsander.'
Clearance of galactose from the blood is performed mai nly
by the liver'· and this function has been shown to be redoxsensitive. Four stages are involved in the conversion of
galactose to glucose in the liver:
(1) galactose + ATP --7 galactose-I-phosphate + ADP ;
(2) galactose-I-phosphate + UTP --7 UDP-galactose +
pyrophosphate;
(3) UDP-galactose --7 UDP-glucose;
(4) UDP-glucose + pyrophosphate --7 glucose-I-phosphate + UTP.
Reaction 3, catalysed by UDp-galactose-4-epimerase, is
NAD-dependent and is inhibited by NADH2 ."·1l Robin son
et al." showed that the r atio of oxidized to reduced NAD
rather than the absolute amounts of these nucleotides was
important with regard to inhibition. Under reducing conditions, therefore, reaction 3 becomes rate-limiting for the
conversion of galactose to glucose and hence for removal
of administered galactose from the blood. This is well
illustrated by the effect of alcohol which has been shown
to inhibit oxidation of galactose in vitro· and the elimination of administered galactose in vivo."·14
S. Afr. Med. J., 46, 772 (1972).
The redox state within cytoplasmic and mitochondrial compartments of the liver cell in symptomatic porphyria (porphyria cutanea tarda symptomatica, Pcf) is of considerable interest with regard to the pathogenesis of this disease.
In PCf there is hepatic accumulation and urinary excretion of higher carboxylated porphyrins. Heikel et af:
suggested that the excessive porphyrins in the liver in cutaneous porphyria may originate from porphyrinogens
which have 'escaped' from the haem biosynthetic pathway.
Porphyrinogens, the true intermediates in haem biosynthesis, are highly unstable compounds and can readily undergo autoxidation to porphyrins. Reducing conditions presumably play a part within the cell in preventing this irreversible oxidation. Goldberg and Rimington' further proposed that in the porphyric liver the cytoplasmic redox
state, as reflected by NAD/NADH2 and NADP/NADPH,
ratios, may be sufficiently altered to promote excessive
oxidation of porphyrinogens.
On the other hand Labbe' has related the intramitochondrial redox state to porphyrinogenesis. His hypothesis
is based on the observation that many porphyrinogenic
substances are inhibitors of NADH., oxidase. He proposed
that this action leads to induction of an intramitochondrial
NADHo-dependent fumarate reductase followed in turn
by substrate induction of succinyl CoA synthetase and
5-aminolaevulinate (ALA) synthetase, the rate-limiting
enzyme for liver haem biosynthesis" If such a sequence
of events does in fact occur in porphyric liver it could
provide an explanation for the well known aggravating
effect of ethanol in Pcf. Oxidation of ethanol in
the liver characteristically leads to incn;ased NADH.,/
NAD ratios.5 . ' In the present study a si mple indirect estimate of the NAD/NADH2 ratio of the liver cell
cytoplasm in patients with symptomatic porphyria was
attempted.
* D ate
received:
IO Augus t 1911.
MATERIALS AND METHODS
Four fem ale and 7 male Bantu patients with clinical and
biochemical evidence of symptomatic porphyria participated voluntarily in the study. All were inpatients at King
Edward VIII Hospital, Durban, and did not have access
to alcohol.
Patients were fasted overnight and a percutaneous live r
biopsy was performed the following morning under local
anaesthesia with a Menghini needle. Hepatic ALA synthetase activity was determined by micro-assay according to
Z ai l and J oubert." Histological examination was carried
out on a portion of the biopsy specimen in those cases
S.-A.
10 Junie 1972
MEDIESE
773
TYDSKRIF
(Byvoegsel-Suid-Afrikaanse Tydskrif vir Laboratorillm- en KlinieklVerk)
where it was required for diagnostic purposes. Immediately after the liver biopsy an intravenous galactose tolerance
test was performed. A battery of standard 'liver function
tests' was also carried out in each case. Porphyrins and
porphyrin precursors were determined in casual urine and
stool specimens by methods cited previously." Alcohol was
administered to 3 volunteers prior to liver biopsy. Commencing in mid-morning on the preceding day, each consumed 300 ml of 96% (v/v) ethanol suitably diluted with
water and soft drinks over a period of 8 hours. After an
overnight fast. each consumed a further 150 ml within I - 2
hours. Liver biopsy was performed 3 - 4 hours later.
Intravenous Galactose Tolerance Test
The single-dose intravenous galactose tolerance test described by Tengstrom" was employed. Galactose was obtained from A. B. Kabi, Stockholm, as a sterile 30% solution containing not more than 0,3 % glucose and not more
than 1,5% of carbohydrates other than galactose.
Patients, prepared as described above, received an intravenous infusion of 350 mg galactose per kg body
weight over a period of I - 3 minutes. Venous blood was
collected at IQ-minute intervals for 60 minutes and 0,1 ml
aliquots were transferred immediately to tubes containing
2 ml of 0,025N NaOH to prevent further metabolism of
galactose. After neutralization with ZnSO., galactose was
determined in the supernatant by an enzymatic method."
Values thus obtained were plotted against time on semilogarithmic paper and the Tt for galactose was determined graphically from the straight line best fitting the
curve. 1T
RESULTS
The clinical and biochemical details of the patients studied
appear in Table I. The results of the galactose tolerance
tests, ALA synthetase assays and liver function tests are
shown in Table H.
In normal subjects Tengstrom 17 found the mean galactose
Tt to be 12,0 minutes (SD 2,6) and he therefore considered the upper limit of the normal range to be 17
minutes. By these criteria, all but 1 of the values obtained
in the present study were abnormal. Galactose Tt ranged
from 17 to 54 minutes, with a mean value for the 11
patients of 29,8 minutes. In most cases liver ALA synthetase activity was moderately increased but in the 3 patients
who received ethanol prior to biopsy, the activity was
markedly elevated. Statistical analysis of these results
according to Bailey'· did not reveal a significant correlation between galactose Tt and ALA synthetase activity
at the 5% level; r was found to be 0,664 as opposed to
an expected value of 0,707. Likewise it is apparent that
there is no significant correlation between galactose Tt
and urinary porphyrin excretion.
Results of the liver function tests show evidence of
hepatocellular dysfunction consistent with the histological
findings detailed in Table I. Hyperbilirubinaemia was
noted in only 3 patients, whereas all cases showed inversion of the albumin: globulin ratio and slight to moderate
elevation of serum transaminase levels.
DISCUSSION
There is good evidence that symptomatic porphyria, like
the genetic varieties of hepatic porphyria, is also an 'overproduction disease' in that increased activity of liver ALA
synthetase and indications of an enlarged endogenous ALA
pool have been found in this condition."'''' Although it is
known that alcohol'" and other substances can cause increased hepatic ALA synthetase activity, the mechanism
of induction is unknown. The finding of no significant
correlation between galactose Tt and hepatic ALA synthetase activity or urinary porphyrin excretion does not
support the hypothesis that the induction of this enzyme
TABLE I. CLINICAL AND BIOCHEMICAL DETAILS OF PATIENTS
WITH
SYMPTOMATIC PORPHYRIA
Stool (dry wt)
Urine
Case
No.
Age
Sex
1
2
3
4
5
6
49
68
69
42
<9
40
F
F
F
F
M
M
7
8
65
40
M
M
9
10
11
42
60
30
M
M
M
+ =
positive screen test.
Liver histology
Uroporphyrin
(fJ.g/100 ml)
Corproporphyrin
(fJ.g/g)
Protoporphyrin
(fJ.g/g)
I
138
300
63
146
1 131
710
91
172
112
14
62
52
78
<1
<1
573
740
79
38
10
<1
<1
<1
409
123
252
250
30
18
32
39
2
ALA
(fJ.g/ml )
PBG
(fJ.g/ml)
<1,0
0
143
5,2
8,3
1,6
<1
<1
<1
3,6
2,0
3,5
1,2
1,0
T
Cirrhosis and marked siderosis.
Cirrhosis and marked siderosis.
Portal fibrosis, marked siderosis.
Liver cell damage, portal fibrosis,
moderate siderosis.
'Active
rosis.
cirrhosis',
moderate side-
Cirrhosis, moderate siderosis.
LKW 59
+
17
S.A.
774
MEDICAL
10 June 1972
JOURNAL
(Supplement-South African Journal of Laboratory and Clinical Medicine)
LCM 60
TABLE 11. GALACTOSE T1f2, LIVER ALA SYNTHETASE ACTIVITY AND 'LIVER FUNCTION TESTS' IN PATIENTS WITH
SYMPTOMATIC PORPHYRIA
Serum
Prothrombin
index
(min)
ALA synthetase
activity flmoles
ALA formed/mg
total liver P/hour
33,5
23,0
36,5
17,0
21,0
19,0
35,0
27,5
32,0
54,0
29,0
29,8
10,6
1,7
18,4
8,8
11,9
0
12,1
8,6
85,8
50,8
37,5
22,4
94
100
100
86
100
Galactose
Case
No.
1
2
3
4
5
6
7
8
9'
10'
11'
Mean
TV2
%
99
80
91
71
85
82
Bilirubin
(mg/l00 ml)
0,9
0,7
3,4
1,2
0,6
0,5
0,7
1,7
0,9
0,9
1,7
1,2
Alkaline
phosphatase
(KA units)
9
14
16
4
15
15
17
9
10
7
31
13
Transaminases
Albumin
(g/100 ml)
Globulin
(g/100 ml)
3,2
2,9
1,9
2,5
3,3
2,0
1,5
3,7
3,2
2,4
2,9
2,7
5,2
5,7
6,0
5,6
5,0
7,0
5,9
4,3
4,4
5,0
6,2
5,5
Alanine
Aspartate
(Karmen units)
137
168
47
56
68
54
86
77
68
65
104
69
54
28
51
14
63
61
121
27
74
60
.. Received ethanol.
may be a consequence of an alteration in the redox state
of the adenine nucleotides in the cytoplasmic compartment of the liver cell. However, it is probable that galactose Tt in these patients is not a reliable measure of
NAD/NADH., in the liver cell sap, but is rather an index
of hepatocellular dysfunction, as are the elevated serum
transaminase levels.
Galactose tolerance is a measure of the redox state of
the liver cell only as long as reaction 3 remains ratelimiting for the conversion of galactose to glucose. Under
pathological conditions some other factor, e.g. hepatic
blood flow, may become crucial. It has been shown that
liver circulation is decreased by approximately one third
in cirrhosis and that this reduction is associated with a
marked fall (about 50% in hepatic bromsulphthalein extraction).~2 Hence, abnormal galactose tolerance in cirrhotic patients",H may, similarly, be attributable to diminished
liver circulation. Since cirrhosis or some degree of hepatocellular pathology was present in all 11 patients, it is
quite likely that diminished hepatic blood flow contributed
significantly to the abnormal values for galactose Tt
obtained in the majority of subjects in the present study.
Another factor meriting consideration is the concentration in the liver of ATP, the nucleotide which is required
for reaction 1 in the sequence of reactions converting galactose to glucose. O'Donnel et al." found decreased hepatic
levels of ATP in patients with various types of hepatocellular disease. Consequently, it is quite conceivable that
in some patients with symptomatic porphyria, reaction 1,
and not reaction 3, is the rate-limiting one. The fact that
in 2 of the 3 patients who received ethanol galactose, Tt
was close to the mean value for the 11 patients, accords
with the finding of Stenstam" that alcohol administered to
patients with liver disease does not further impair oral
galactose tolerance and supports the above explanation,
since there is no doubt that ethanol affects the cytoplasmic
NAD/NADH~ ratio in the hepatocyte.""
Other means will, therefore, have to be employed in
order to assess the redox state of the liver cell in symptomatic porphyria. This information remains of prime
importance in understanding the pathogenesis of this
disease. It is of considerable interest that hexachlorobenzene, which can produce a porphyric state in man closely
resembling PCT," has recently been shown to be capable
of profoundly influencing hepatic NAD/NADH~ ratios in
animals."
This work was supported by a grant from the South African
Medical Research Council.
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