Download Nerve function and dysfunction in acute intermittent porphyria

doi:10.1093/brain/awn152
Brain (2008), 131, 2510 ^2519
Nerve function and dysfunction in acute
intermittent porphyria
Cindy S.-Y. Lin,1,2,3 Arun V. Krishnan,1,2 Ming-Jen Lee,4 Alessandro S. Zagami,1 Hui-Ling You,4
Chih-Chao Yang,4 Hugh Bostock5 and Matthew C. Kiernan1,2
1
Prince of Wales Clinical School, 2Prince of Wales Medical Research Institute, 3School of Medical Sciences, University of New
South Wales, Randwick, Sydney, New South Wales, Australia, 4Department of Neurology, National Taiwan University
Hospital, Taipei, Taiwan and 5Sobell Department of Motor Neuroscience and Movement Disorders, Institute of Neurology,
University College London, Queen Square, London, UK
Correspondence to: Matthew C. Kiernan, Associate Professor, Prince of Wales Medical Research Institute, Barker Street,
Randwick, Sydney, New South Wales 2031, Australia
E-mail: [email protected]
Acute intermittent porphyria (AIP) is a rare metabolic disorder characterized by mutations of the porphobilinogen deaminase gene. Clinical manifestations of AIP are caused by the neurotoxic effects of increased porphyrin
precursors, although the underlying pathophysiology of porphyric neuropathy remains unclear. To further investigate the neurotoxic effect of porphyrins, excitability measurements (stimulus-response, threshold electrotonus, current^threshold relationship and recovery cycle) of peripheral motor axons were undertaken in 20 AIP
subjects combined with the results of genetic screening, biochemical and conventional nerve conduction studies.
Compared with controls, excitability measurements from five latent AIP patients were normal, while 13 patients
who experienced acute porphyric episodes without clinical neuropathy (AIPWN) showed clear differences in
their responses to hyperpolarizing currents (e.g. reduced hyperpolarizing I/V slope, P _ 0.01). Subsequent mathematical simulation using a model of human axons indicated that this change could be modelled by a reduction
in the hyperpolarization-activated, cyclic nucleotide-dependent current (IH). In contrast, in one patient tested
during an acute neuropathic episode, axons of high threshold with reduced superexcitability, consistent with
membrane depolarization and reminiscent of ischemic changes. It is proposed that porphyrin neurotoxicity
causes a subclinical reduction in IH in AIPWN axons, whereas porphyric neuropathy may develop when reduced
activity of the Na+/K+ pump results in membrane depolarization.
Keywords: porphyria; haem; nerve excitability; inward rectification (IH); ischaemia
Abbreviations: AIPWN = acute intermittent porphyria without neuropathy; AIPN = acute intermittent prophyric
neuropathy; ALA = -aminolevulanic acid; ATP = adenosine triphosphate; APB = abductor pollicis brevis;
CMAP = compound muscle action potential; Cr = creatinine; GBS = Guillain Barre¤ syndrome; GH = inward rectifying
conductance; HMBS = hydroxymethylbilane synthase gene; IH = hyperpolarizing activated conductance/inward rectification;
PBGD = porphobilinogen deaminase; PN = porphyric neuropathy; TA = tibialis anterior; TE = threshold electrotonus
Received November 26, 2007. Revised May 8, 2008. Accepted June 20, 2008. Advance Access publication July 17, 2008
Introduction
Porphyrins (derived from the ancient Greek word porphura,
purple pigment) are the main precursors of heme, an
essential constituent of haemoglobin, myoglobin, catalase,
perioxidase, respiratory and P450 liver cytochromes.
Human porphyrias are inherited metabolic disorders
resulting from deficiency of the enzymes involved in the
biosynthesis of heme, the principal product of porphyrin
metabolism. Of these, acute intermittent porphyria (AIP),
an autosomal dominant inherited disorder, represents the
most common form of the neuroporphyrias.
AIP is characterized by deficiency of porphobilinogen
deaminase (PBGD), the third enzyme in the porphyrin–
heme biosynthetic pathway, which occurs due to mutations
in the hydroxymethylbilane synthase gene (HMBS). To
date, over 300 different mutations within PBGD have been
identified (Hrdinka et al., 2006). Acute episodes are
biochemically characterized by overproduction and tissue
ß The Author (2008). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: [email protected]
Excitability properties of acute intermittent porphyria
accumulation of the porphyrin precursors, deltaaminolevulinic acid (ALA) and PBG.
Clinical expression of AIP is highly variable and up to
90% of heterozygotes may remain asymptomatic throughout life. In those clinically affected, acute episodes usually
manifest after puberty, more commonly in females. AIP
symptoms comprise a variety of neurological (severe pain,
sensory loss, motor weakness, convulsions) and more
general manifestations (fever, tachycardia, nausea, vomiting,
constipation), as well as respiratory failure that can lead to
coma and death (Kauppinen, 2005). Porphyria has also
been linked to the madness that affected King George III of
Great Britain (Cox et al., 2005) and the illness of Vincent
van Gogh (Arnold, 2004). Symptoms of AIP do not
generally develop unless a person with the enzymatic
deficiency encounters a trigger such as hormonal substances, medications, sun exposure or dietary factors
(Herrick and McColl, 2005).
Porphyric neuropathy (PN) typically presents as a motor
neuropathy of the axonal type (Albers and Fink, 2004). The
pathophysiology underlying axonal dysfunction in AIP
remains unknown. Heme is an essential component of
the mitochondrial electron transport chain and critical to
aerobic metabolism and ATP production. Fast axonal
transport is highly energy dependent, and diminished
ATP availability would disrupt this process. The accumulation of neurotoxic heme precursors may lead to impaired
function of the axonal Na+/K+ pump with consequent
alteration in membrane potential to cause neuronal cell
death and axonal degeneration (Albers and Fink, 2004).
Changes in axonal ion channel function and membrane
potential may now be investigated in vivo using novel nerve
excitability methods, recently adapted for clinical use
(Kiernan et al., 2000, 2001b, 2004). In the present study,
multiple excitability parameters were recorded to investigate
axonal properties in patients with AIP, and also in
asymptomatic carriers of genetic mutations linked to AIP.
The aim was to test the hypothesis that the development of
porphyric neuropathy may be linked to axonal membrane
depolarization.
Methods
Clinical assessment and neurophysiological investigations including nerve conduction studies (NCS) and nerve excitability studies
were undertaken in 15 AIP patients (10 with previous clinical
episodes and confirmed genetic mutations; 5 with previously
confirmed clinical episodes without detected mutation) and 5
asymptomatic latent gene carriers. The clinical and biochemical
characteristics of this cohort are summarized in Table 1. Clinical
diagnosis was confirmed through assessment of serum biochemistry indices, enzyme activity and mutation screening of patients
and their families. Patients were recruited from both the Prince
of Wales Hospital and the National Taiwan University Hospital.
All subjects gave informed consent to the procedures, which
were approved by the ethics committee of National Taiwan
University Hospital and Human Research Ethics Committees of
Brain (2008), 131, 2510 ^2519
2511
the University of New South Wales and the South Eastern Sydney
Area Health Service Human Research Ethics Committee.
The studies were performed in accordance with the Declaration
of Helsinki.
Routine nerve conduction studies used conventional techniques
(Kimura, 1983). Excitability studies were undertaken on the
median and peroneal nerves. Compound muscle action potentials
(CMAPs) were recorded using surface electrodes from the
abductor pollicis brevis (APB) and tibialis anterior (TA) with
the active electrode at the motor point and the reference electrode
4 cm distal (Eduardo and Burke, 1988). The signal was amplified,
filtered (3 Hz–3 kHz) and digitized by computer (PC) with an
analog-to-digital board (National Instrument DAQCard for
PCMCIA). Stimulus current was applied via non-polarizable
electrodes with the cathode over the median nerve at the wrist
and the anode 10 cm proximal over muscle. Stimulus waveforms
were generated by the computer and converted to current by an
isolated linear bipolar constant-current source (maximal output
50 mA). Stimulation and recording were controlled by software
(QTRAC version 8.2, ßInstitute of Neurology, London). Motor
axons were studied using the multiple excitability protocol,
TRONDCM (Z’Graggen et al., 2006). Test current pulses of
1-ms duration were applied to produce a target potential that
was on the fast-rising phase of the stimulus–response curve, 40%
of the maximal CMAP. These stimuli were combined with suprathreshold conditioning stimuli or subthreshold polarizing
currents. The amplitude of the CMAP was measured from
baseline to the negative peak. The automated excitability protocols
incorporated the following measures:
(i) ‘Stimulus-response curves’ recorded the response as the
stimulus was increased from zero to where it produced
supramaximal potentials.
(ii) Threshold was then recorded using a 1-ms test stimuli
applied 200 ms after the onset of a long-lasting subthreshold
polarizing current, the strength of which was altered in 10%
steps from +50% (depolarizing) to 100% (hyperpolarizing)
of the control threshold. This produced a ‘current–threshold
relationship’ (I/V), analogous to the current–voltage relationship that depends on the rectifying properties of the
nodal and internodal axolemma. The slope of this curve may
be used to provide an estimate of the threshold analog of
input conductance (Kiernan and Bostock, 2000). Values
of the hyperpolarizing I/V slope were derived from the
slope of the most hyperpolarized three points of the I/V
curve (Kiernan et al., 2001a) as depicted in Fig. 1B.
(iii) Changes in threshold associated with electrotonic changes in
membrane potential, termed ‘threshold electrotonus’ (TE),
have a time course similar to the underlying changes in
membrane potential (Bostock and Baker, 1988; Bostock
et al., 1998; Kiernan et al., 2005a). Prolonged subthreshold
currents were used to alter the potential difference across the
internodal axonal membrane. The subthreshold polarizing
currents were of 100-ms duration and set to be +40%
(depolarizing, TEd) and 40% (hyperpolarizing, TEh) of the
control threshold current (i.e. the current required to
produce the unconditioned target CMAP, which was
40% of maximum). Threshold was tested at different
time points during and after the 100-ms polarizing currents.
(iv) The final part of the protocol measured the recovery of
axonal excitability following delivery of a supramaximal
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conditioning stimulus with conditioning-test intervals from
2 to 200 ms. The ‘recovery cycle’ of excitability consisted
of refractory period at short conditioning-test intervals due
to inactivation of transient Na+ channels with a resultant
increase in threshold. This was followed by a period of
superexcitability due to the depolarizing afterpotential
(Barrett and Barrett, 1982; Blight, 1985; Bowe et al., 1987).
The late-subexcitable period during which axonal excitability
was reduced reflected the kinetics of voltage-dependent slow
K+ channels activated by the conditioning stimulus (Baker
et al., 1987; Stys and Waxman, 1994; Kiernan and Bostock,
2000; Schwarz et al., 2006).
Electrical model of nerve excitability
To model the excitability changes in human motor axons induced
by porphyria, mathematical simulations were undertaken using a
model of the human axon (Bostock et al., 1991, 1995; Kiernan
et al., 2005b, c; Jankelowitz et al., 2007). Transient Na+ channels
were modelled using the voltage clamp data (Schwarz et al., 1995),
and persistent Na+ currents were added (Bostock and Rothwell,
1997). The equations for a single node and internode, representing
a spatially uniform axon, were evaluated by integration over
successive small time steps (Van Euler’s method, Press et al.,
1992), using a maximal integration interval of 3 ms. At times
corresponding to those in human nerve excitability recordings, the
excitability of the model nerve was tested repeatedly to determine
threshold with an accuracy of 0.5%. The discrepancy between the
thresholds determined for the model and those determined from a
sample of real nerves was scored as the weighted sum of the error
terms: [(xm xn)/sn]2, where xm is the threshold of the model,
xn the mean and sn the standard deviation of the thresholds for
the real nerves. The weights were the same for all threshold
measurements of the same type (e.g. recovery cycle), and chosen
to give an equal total weight to the different types of threshold
measurement: current/threshold relationship, threshold electrotonus and the recovery cycle. The standard model was obtained by
minimizing the discrepancy between the model and the normal
control data with an iterative least squares procedure, so that
alteration of any of the above parameters would make the
discrepancy worse.
Statistical analysis
Values obtained in this study were compared to established
normative data for NCS for this unit (Burke et al., 1974) and
elsewhere (Ma and Liveson, 1983). For the excitability studies, a
separate group of normal Taiwanese controls (11 healthy females,
2 males) was recruited to match the gender ratio of the AIP
patients (ages: AIPWN 19–54 years, mean 33.1 years; controls
19–58 years, mean 37.7 years). Recordings were compared using
the Student’s t test. All data are expressed as mean SE, and
P-values50.05 were regarded as statistically significant.
Results
Demographic, biochemical and genetic data for the 20
subjects are detailed in Table 1. After clinical and excitability
testing, one patient was found to be diabetic and his data
were excluded from the analysis given that diabetes
mellitus may affect axonal excitability (Kitano et al., 2004;
C. S.-Y. Lin et al.
Table 1 Summary of clinical and biochemical data from AIP
patients (either clinically affected or latent gene carrier)
Patient # Age Sex Clinical episodes PBGD Mutation identified
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
25
42
36
35
30
50
54
34
19
24
25
22
27
69
9
23
18
47
54
38
F
F
F
F
F
F
F
F
F
F
M
M
F
M
F
F
M
M
F
F
+
+
+
+
+
+
+
+
+
+
+
+
+
+/DM
+
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
N
N
N
N
Y
Y
Y
Y
Y
NT
PBGD mutation screening was undertaken in most patients, and
the mean PBG level for 13 symptomatic patients was 525 nm/h/
mlRBC (normal 4 30). Patient #14 was excluded from the subsequent data analysis due to presence of co-existent diabetes mellitus
(DM). Patient #20 developed acute porphyric neuropathy.
Krishnan and Kiernan, 2005). None of the other subjects had
a history of other medical conditions known to affect nerve
function other than AIP.
The results presented in this study were categorized
according to three groups: (i) 13 patients with AIP without
neuropathy (AIPWN); the diagnosis of porphyria was
confirmed by elevated ALA levels, positive porphyrin
urine tests and in 10 cases a confirmed PBGD mutation;
however, each of these patients had normal NCS, further
confirming that they did not have neuropathy; (ii) five
asymptomatic latent AIP gene carriers; (iii) one patient
with AIP who developed clinical neuropathy (AIPN) was
studied during an acute neuropathic relapse.
Multiple excitability measurements
in AIPWN
Comparison of mean data from AIPWN patients with
control subjects established no significant difference in
the stimulus–response curves (Fig. 1A). During threshold
electrotonus (Fig. 1C), the threshold changes to the
depolarizing conditioning currents were similar. However,
AIPWN patients had greater threshold changes to hyperpolarizing conditioning currents. This became more apparent in the hyperpolarizing part of the current threshold
(I/V) relationship (Fig. 1B), which showed that the
threshold increase induced by strong hyperpolarizing
currents of 200 ms duration in AIPWN was significantly
greater than that in control subjects. The I/V relationship
Excitability properties of acute intermittent porphyria
Brain (2008), 131, 2510 ^2519
2513
Fig. 1 Excitability findings from patients who had previously experienced porphyric attacks (n = 13) without evidence of neuropathy
(AIPWN). Mean data (SE) of excitability parameters recorded from AIPWN patients (filled circles) and normal controls (n = 13, open
circles). (A) Stimulus-response curves; (B) Current^voltage (I/V) relationship demonstrating outward and inward rectification and the
hyperpolarizing I/V slope was calculated with the three most hyperpolarizing points; (C) Threshold electrotonus; (D) Recovery cycle;
no significant difference found in refractoriness, superexcitability and subexcitability.
reflects the rectifying properties of the axon, both
nodal and internodal (Baker et al., 1987). The steeper
the curve (i.e. the greater the current required to produce a change in threshold) the higher the input conductance. The steepening towards the top indicates outward
rectification, an accommodative response to the depolarizing current associated with the activation of fast and slow
K+ channels. In contrast, the steepening of the curve
towards the bottom represents inward rectification, an
accommodation to the hyperpolarizing current due to
an increase in the hyperpolarization-activated conductance,
IH (Pape, 1996). These conductance properties are reflected
by the slope of the I/V curves, shown in Fig. 2A:
the minimum I/V slope was lower in AIPWN than in
NC (AIPWN: 0.195 0.004; NC: 0.232 0.009; P50.005),
and the hyperpolarizing I/V slope, calculated from the
most hyperpolarized three points of the I/V curve
(Kiernan et al., 2001a), was significantly reduced
(AIPWN: 0.276 0.017, controls: 0.350 0.016; Fig. 2B,
P50.01).
The findings for the recovery cycle in AIPWN are
depicted in Fig. 1D. There were no significant differences
between AIPWN and control subjects for the excitability
parameters
derived
from
their
recovery
cycles
Fig. 2 Changes in the slope of the current^ threshold (I/V)
relationship: (A) The slopes of the I/V relationship (derived from
Fig. 1B), mean data of AIPWN patients (filled circles) superimposed
on normal controls (open circles). No difference was shown in
the depolarizing direction, in contrast to the significant difference
established with greater hyperpolarization. (B) Histograms of
the hyperpolarizing I/V slope: the slope is significantly reduced
(P50.01) in AIPWN patients (SE) (filled) in comparison to
normal controls (open) indicating a reduction in the expression
of inward-rectifying conductances (IH).
(refractoriness, AIPWN: 44.4 10.3%, controls: 28.7 5.8%; superexcitability, AIPWN: 24.1 1.63%, controls:
25.7 1.53%; subexcitability, AIPWN: 14 1.59%, controls: 15.5 1.44%).
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Brain (2008), 131, 2510 ^2519
Of the 13 clinically affected patients with AIPWN, 10 had
confirmed PBGD gene mutations, and subgroup analyses
of these 10 again confirmed significant differences in the
minimum and hyperpolarizing I/V slopes (P50.05).
To investigate whether these changes in nerve excitability
occurred independently of clinical episodes, excitability
studies were also undertaken in a group of asymptomatic
subjects who expressed PBGD genetic mutations linked to
the development of porphyria. Findings in these asymptomatic carriers were almost identical to normal controls
(P40.05 for all parameters).
Reproducibility
To investigate the reproducibility of these excitability changes,
repeat testing was undertaken on patient #1 on seven different
occasions. This series of studies confirmed that excitability
parameters were highly repeatable, with small variability
between multiple sessions of testing (Fig. 3). The stimulus–
response curve of this patient was consistently normal
(Fig. 3A), but the I/V relationship (Fig. 3B) showed a consistent abnormality, similar to that of the grouped patient data
(Fig. 1B). Thus, although the mean minimum I/V slope (0.19)
was within the range of normal control values (0.185–0.29),
it was consistently below the normal mean of 0.23
(P50.001). The consistency of the repeated superexcitability
C. S.-Y. Lin et al.
measurements in patient #1 (Fig. 3D), which had a standard deviation of 1.90%, is relevant to the interpretation
of the observations on patient #20, described in the next
section. Previous data on intra-individual variation in
superexcitability in normal subjects (Kiernan et al., 2000)
indicated a similar standard deviation of 1.97%.
Acute porphyric neuropathy during a relapse
Patient #20, a previously undiagnosed 38-year-old female,
presented acutely with a 3-day history of generalized
weakness, manifested by difficulty in walking up stairs
and also in raising her arms. In addition, the patient complained of intermittent abdominal pain and change in urine
colour (Fig. 4). Cranial nerve examination revealed a lower
motor neuron pattern of facial weakness bilaterally. Limb
power was initially graded at Medical Research Council
grade 4/5 in most groups and progressed to grade 3/5 over 3
days. Deep tendon reflexes remained symmetrically normal
and plantar responses were flexor. Sensory examination was
normal.
In this acutely symptomatic patient, nerve conduction
studies demonstrated reductions in compound motor
action potential (CMAP) amplitudes, with relative preservation of conduction velocities, consistent with a
motor neuropathy of the axonal type (Table 2). Sensory
Fig. 3 Reproducibility of excitability recordings in a single AIPWN patient (patient #1, Table 1). This patient had several porphyric attacks
and seven repeated excitability studies were undertaken on different days (mean SE; filled circle), superimposed with normal controls
(mean SE; open circle).
Excitability properties of acute intermittent porphyria
values were within normal limits as were proximal H-reflex
and F-wave parameters. Electromyography demonstrated
widespread reduction in interference patterns in upper and
lower limb muscles, with minor evidence of spontaneous
activity, limited to the right deltoid.
Biochemical testing demonstrated an increase in plasma
porphyrin levels and elevations in the ratios of urinary
Fig. 4 Urine sample obtained from patient #20 during the porphyric attack, with dark port wine colour evident after the sample
was left standing in sunlight, in comparison to a control sample.
Table 2 Nerve conduction parameters from patient #20,
who presented with acute neuropathy
R sural
R radial
R tibial (to AH): ankle
R deep peroneal
(to EDB): ankle
fibular neck
L deep peroneal
(to EDB): ankle
R median (to APB): wrist
R ulnar (to ADM): wrist
R soleus H reflex
F-waves R. AH
Amplitude
Conduction velocity/
distal motor latency (m/s)
21.5 mV
48 mV
5.6 mV
2.8 mV
41
53
5.1
4.2
2.3 mV
0.9 mV
42
4.3
3.8 mV
4.3 mV
3.8
2.9
31.7
50.9^53.7
(100% persistence)
NCS established reduction in motor amplitudes, most prominent
for left EDB, right EDB and right APB. Sensory amplitudes were
within normal limits as were proximal H-reflex and F-wave
parameters. EDB = extensor digitorum brevis; AH = abductor hallucis; APB = abductor pollicis brevis; ADM = abductor digiti minimi.
Brain (2008), 131, 2510 ^2519
2515
-aminolevulanic acid (ALA) and porphobilinogen (PBG)
to creatinine ratio, consistent with AIP. Treatment with a
high glucose diet (400 g daily) and infusions of heme
arginate (150 mg daily) led to reduction in plasma porphyrin indices, associated with gradual clinical improvement.
In this symptomatic acute porphyric neuropathy patient,
excitability studies established that axons were of high
threshold in both upper (median nerve) and lower limb
(peroneal nerve) recordings, with stimulus–response curves
shifted to the right, and rheobase increased (median nerve
6.6 mA, normal 1.3–5.0 mA; peroneal nerve 8.8 mA,
normal 1.5–6.0 mA).
Recordings on this patient were repeated 4 months after
the initial presentation when the patient had clinically
improved and demonstrated significant improvement in
stimulus–response measures. Furthermore, superexcitability,
a sensitive marker of membrane potential (Kiernan and
Bostock, 2000), demonstrated marked improvement and
had increased from 17.7% during the acute phase to 31.0%
during the recovery phase (Fig. 5). Although the value of
17.7% is within the 95% confidence limits for superexcitability in the control group (14.7–36.7%), because of
the normal intra-individual consistency of this measure, as
indicated by the repeatability study on patient #1, the
difference of 12.3% between the two recordings from
patient #20 in Fig. 5 is statistically significant (P50.0001,
estimated on the basis of the above SD of 1.9%). In light of
the established reduction of superexcitability with membrane depolarization (e.g. Kiernan and Bostock, 2000), the
results in Fig. 5 suggest that there is axonal depolarization
during the acute porphyric relapse, with subsequent
normalization of membrane potential during the period
of clinical recovery. The improvement in clinical symptoms
and nerve excitability findings was reflected in biochemical
markers of disease activity, with a marked improvement
in the urinary porphobilinogen/creatinine ratio (PBG/Cr)
Fig. 5 Recovery cycles of acute phase of the illness (open circle)
and after glucose and heme treatment (filled circle) demonstrated
improvement after treatment, particularly an increase in superexcitability (block arrow).
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Brain (2008), 131, 2510 ^2519
Fig. 6 Fitting a standard mathematical model to the excitability
changes recorded from 13 AIPWN patients. Left: Threshold electrotonus; Right: Current^ threshold relationship. Grey lines: model
fitted to control group (open circles in Fig. 1); black lines: model
fitted to AIPWN recordings (filled circles in Fig. 1) by 27% reduction in IH . Recovery cycles were unaffected by change in IH.
during the recovery period (acute phase, 80 mol/mmol:
recovery phase, 39 mol/mmol).
Mathematical simulations of abnormal
excitability properties in AIPWN
To clarify the changes in nerve excitability induced by
porphyria, the mathematical model of a human motor axon
was first adjusted to provide a close match to the recordings from the control group. The model was used to
explore whether changes in any membrane parameter could
reproduce the changes seen in the AIPWN patients. The
best fit was obtained by reducing the inward rectifying
conductances (GH) by 27%, which reduced the discrepancy
by 61%. This fitting procedure, which takes into account
the effects of conductances on threshold electrotonus,
current threshold relationships and the recovery cycle,
supported the interpretation that inward rectifying conductance (IH) was reduced in AIPWN patients (Fig. 6).
Discussion
The present study has combined clinical, biochemical and
genetic findings with assessment of conventional neurophysiological and nerve excitability parameters across a
spectrum of AIP patients and asymptomatic gene mutation
carriers. Nerve excitability properties were normal in
asymptomatic AIP patients, whereas AIPWN patients who
suffered porphyric attacks demonstrated consistent differences in their responses to hyperpolarizing currents. These
abnormalities in nerve excitability were significant and
reproducible in a symptomatic patient who underwent
repeat studies on seven different occasions.
In contrast, in a patient who presented with weakness
and abdominal pain due to AIP complicated by neuropathy, peripheral axons in the upper and lower limbs were of
high threshold, and nerve excitability findings were consistent with axonal membrane depolarization. Reduction in
C. S.-Y. Lin et al.
the levels of heme precursors induced by treatment with a
high glucose diet and heme arginate led to significant
clinical improvement and normalization of nerve excitability parameters. This combination of changes in nerve
excitability was not predicted, but is reminiscent of
apparently contradictory changes previously described in
diabetic neuropathy. Thus, Horn et al. (1996), in an early
clinical study of threshold electrotonus, found evidence for
reduced inward rectification in motor and sensory axons in
diabetic patients, which was related to age and neuropathy.
More recently, Krishnan and Kiernan (2005) have found
evidence of membrane depolarization in patients with
established diabetic neuropathy. Mathematical modelling of
the changes in axonal excitability has supported the interpretation that the changes are associated with a reduction
in the hyperpolarization-activated current IH.
Mechanisms of neurotoxicity in porphyria
Despite increased understanding of the molecular and
cellular biology of AIP, the mechanisms of neurotoxicity
remain unclear. Previous studies have suggested that up to
40% of patients with porphyria will develop clinical features
of neuropathy (Albers and Fink, 2004). Patients typically
present with predominantly motor involvement, characterized by weakness and areflexia, which may mimic Guillain
Barré syndrome (GBS). Nerve conduction studies are of
prime importance in differentiating these two conditions, as
demonstrated by patient #20 from the present series with
porphyric neuropathy who manifested features of an axonal
neuropathy but lacked the demyelinating features of GBS
(Albers and Fink, 2004) and maintained preserved reflex
function. Nerve excitability abnormalities in this acutely
symptomatic neuropathy patient were consistent with
axonal depolarization and resembled those in ischaemia
(Kiernan and Bostock, 2000). In further comparison,
studies undertaken on patients with axonal GBS have
established that threshold electrotonus (TE) was normal in
acute motor axonal neuropathy (AMAN) (Kuwabara et al.,
2002). Nerve excitability studies may therefore provide the
opportunity to examine neurotoxic mechanisms in greater
detail.
For instance, changes in membrane potential may be
induced by impairment of the axonal Na+/K+ pump
(Kiernan and Bostock, 2000). This pump is electrogenic,
with three Na+ ions being pumped out for every two K+
ions pumped into the axon, leading to a net deficit of
positive charge on the inner aspect of the axonal membrane. Paralysis of the Na+/K+ pump results in the loss
of its electrogenic contribution to the membrane potential and leads to further depolarization due to extracellular accumulation of K+ ions (Kaji and Sumner, 1989; Kaji,
2003). In vitro studies of porphyria provide a theoretical
basis for impairment of Na+/K+ pump function in PN
(Becker et al., 1971, 1975). These previous studies
demonstrated membrane depolarization in muscle fibers
Excitability properties of acute intermittent porphyria
(Becker et al., 1975) and impairment of Na+/K+ pump
function in brain and red blood cells, induced by high
levels of ALA (Becker et al., 1971). It appears that the
blood–nerve barrier does not afford peripheral axons as
good a protection from high levels of ALA in the blood as
that provided to the central nervous system by the blood–
brain barrier (Meyer et al., 1998). Other studies have
suggested that ischaemia, which would also impair Na+/K+
pump function, may play a role in the development of
other complications of porphyria, including abdominal pain
and optic atrophy (DeFrancisco et al., 1979; Lithner, 2000).
Interpretation of excitability changes
in AIPWN patients
The excitability changes in AIPWN patients imply a
reduction in the inward rectifying conductance IH. The
hyperpolarization-activated, cation non-selective (HCN)
channels underlying IH are regulated by neurotransmitters
and metabolic stimuli, so that several mechanisms could
contribute to the change in IH in AIPWN patients. Changes
in the intracellular metabolism acting through changes in
the activity of adenylate cyclase will alter IH (McCormick
and Pape, 1990; Tokimasa and Akasu, 1990). Intracellular
acidosis also shifts the activation curves for IH to the left
(Munsch and Pape, 1999). As mentioned above, IH appears
to be decreased in diabetes mellitus (Horn et al., 1996) and
this has also been found in animal models of diabetes
(Yang et al., 2001), a change that reflects a parallel change
in cAMP and responds to agents that boost intracellular
cAMP (Quasthoff, 1998). More recently, nerve excitability
studies supported by modelling have provided evidence of a
selective reduction in IH, very similar to that in the AIPWN
patients, on the affected side in motor axons of stroke
patients (Jankelowitz et al., 2007). In that case, the change
presumably results from a loss in descending input to the
motoneurons, and was attributed to the consequent lack of
impulse activity in the axons.
In the case of porhyria, as in diabetes, it seems that a likely
common factor underlying both the reduction in IH and
membrane depolarization may be cellular hypoxia. In
porphyria, this may result from impairment of energydependent cellular functions by porphyrin metabolites
(Herrick et al., 1987; Kordac et al., 1989; Peters and
Deacon, 2003). Previous studies have suggested that alterations in IH may occur as an early manifestation of hypoxia
and impaired cellular metabolism (Duchen, 1990). The
reductions in IH noted in AIPWN patients in the current
study, and in diabetics by Horn et al. (1996) may therefore
represent a subclinical manifestation of metabolic deficiency.
The potential effects of more severe energy depletion on
axonal Na+/K+ pump function appear self-evident, given that
50% of axonal energy requirement is consumed by the Na+/
K+ pump. Such a reduction in energy delivery would be
expected to cause axonal Na+/K+ pump dysfunction, with
consequent membrane depolarization, as noted in the
Brain (2008), 131, 2510 ^2519
2517
AIPN patient. Similarly, impaired Na+/K+-pump activity
has been implicated by many studies in diabetic neuropathy
(Krishnan and Kiernan, 2005; Krishnan et al., 2008).
In conclusion, the new evidence for membrane depolarization in an AIP patient with neuropathy supports
previous suggestions that impaired Na+/K+ pump function
is involved in the pathogenesis of the neuropathy. In
contrast, AIPWN patients exhibit a specific deficit in IH,
which may be a subclinical sign of impaired metabolism.
Acknowledgements
C.S-Y.L. was supported by C.J. Martin Fellowship from
National Health and Medical Research Council of Australia.
A.V.K. was supported by grants from the National Health
and Medical Research Council of Australia and the
Australian Association of Neurologists. The authors thank
Dr Ann M. Bacsi for helpful clinical input.
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Appendix
Glossary of specialist terms
Depolarization
When the membrane potential becomes less negative, as
occurs during anoxia or ischaemia.
Electrotonus
Changes in membrane potential evoked by subthreshold
depolarizing or hyperpolarizing current pulses.
Hyperpolarization
When the membrane potential becomes more negative.
Inward rectification
The passing of more current in the inward (as opposed to
the outward direction).
Membrane potential
Voltage difference across the axonal membrane (inside–
outside).
Excitability properties of acute intermittent porphyria
Refractoriness
The decrease in excitability during the relative refractory
period after a nerve impulse. It may be expressed quantitatively as the percentage increase in threshold when a test
stimulus is preceded by a conditioning stimulus at an interval
within the relative refractory period (e.g. 2 ms).
Brain (2008), 131, 2510 ^2519
2519
It may be expressed quantitatively as the maximum
percentage reduction in threshold when a test stimulus is
preceded by a conditioning stimulus at an interval within
the superexcitable period.
TEd
Depolarizing threshold electrotonus.
Relative refractory period
The period between the end of the absolute refractory
period and the start of the superexcitable period, when an
axon may be excited but its threshold is increased.
TEh
Subexcitability
Threshold (current)
A decrease in excitability (increase in threshold) that occurs
after the superexcitability phase.
The stimulus current required to excite a single unit, or to
evoke a compound potential that is a defined fraction of
the maximum.
Hyperpolarizing threshold electrotonus.
Subthreshold
(A stimulus) too weak to trigger an action potential.
Threshold electrotonus
Superexcitability
Threshold changes produced by prolonged subthreshold
depolarizing or hyperpolarizing currents, normally corresponding to the induced change in membrane potential
(i.e. electrotonus).
An increase in excitability (or reduction in threshold
current) commonly observed shortly after a nerve impulse.