Clinical Science (1984) 61, 505-509
505
Hypophosphataemic osteomalacia and myopathy: studies
with nuclear magnetic resonance spectroscopy
R. SMITH, R. J. NEWMAN*?, G. K. RADDAS, M. STOKES
AND
A. YOUNG
NuffieId Orthopedic Centre, *Nuffild Department of Orthopedic Surgery and $Department of Biochemimy,
University of Oxford, Oxford, U.K.
(Received 30 November 1983119 April 1984; accepted 1 May 1984)
summary
1. A patient with familial adult-onset hypophosphataemia, whose myopathy was closely
related to the plasma phosphate concentration,
was investigated by phosphorus nuclear magnetic
resonance spectroscopy (31Pn.m.r.) in vivo of the
right flexor digitorum superficialis muscle.
2. During hypophosphataemia induced by
stopping oral phosphate a significant reduction in
measured muscle strength occurred, but the ratios
of the intramyocellular levels of phosphocreatine
(PCr), adenosine triphosphate (ATP) and inorganic
phosphate (Pi) remained unchanged at rest. During
exercise these levels changed, as did the intramyocellular pH, but they did not differ from the
pattern previously recorded in normal subjects.
3. In four adults with inherited infantile-onset
hypophosphataemia (vitamin D-resistant rickets,
VDRR) without myopathy, the n.m.r. measurements were normal at rest and during exercise.
4. In one patient with inherited hyperphosphataemia (tumoral calcinosis) the resting PCr :
Pi ratio was significantly reduced.
Key words: myopathy, nuclear magnetic resonance
spectroscopy, osteomalacia, phosphate.
Introduction
Proximal myopathy is a prominent feature of
some forms of osteomalacia but the biochemical
Correspondence: Dr Roger Smith, Nuffield
Orthopaedic
Centre,
Headington,
Oxford
OX3 7LD, U.K.
t Present address: University Department of
Orthopaedic Surgery, Western Infirmary, Glasgow,
U.K.
abnormality underlying the muscle weakness is
unclear. Under experimental conditions lack of
vitamin D produces disturbances in muscle physiology independent of changes in plasma calcium
[ 1,2]. Hypophosphataemia is also known to impair
muscle function [3] and myopathy may be a
striking symptom of acquired hypophosphataemic
osteomalacia [4].In contrast, patients with infantile-onset inherited hypophosphataemia (vitamin Dresistant rickets, VDRR) have normal muscle
strength [S]. Recent advances in the technique
of high-resolution phosphorus nuclear magnetic
resonance spectroscopy (31P n.m.r.) now provide
an opportunity for the non-invasive and sequential
measurement of the relative intramyocellular concentrations of the major phosphate-containing
metabolites in vivo [6-81.
This method is based on the interaction between
phosphorus nuclei within tissues when placed in a
homogeneous magnetic field and radio frequency
energy. Signals (resonances) that reflect the intramyocellular levels of phosphocreatine (PCr),
adenosine triphosphate (ATP) and inorganic
phosphate (Pi) can be painlessly recorded within
a few minutes and the intracellular pH can be
derived from the spectrum [9].
This paper describes the combined use of 31P
n.m.r. spectroscopy and measurement of muscle
strength and urinary 3-methylhistidine (3-MeH) '
excretion to study the reversible myopathy associated with hypophosphataemia in a rare form of
adult-onset osteomalacia [ 101. For comparison
the results of similar studies on four patients
with inherited infantile-onset hypophosphataemic
rickets and one subject with inherited hyperphosphataemia (tumoral calcinosis) [ I l l are also
presented.
506
R. Smith et al.
P
Experimental
Patients
Patient X aged 3 2 years (pedigree V9 in [lo])
is a member of a family with dominantly inherited
adult-onset hypophosphataemic osteomalacia who
has been successfully treated with oral phosphate
supplements (2.2 g of inorganic phosphate daily)
since 1973 without recurrence of her bone disease.
Her hypophosphataemic 38-year-old sister Y
(pedigree V3 in [ l o ] ) recently developed pathological rib fractures with radiological and histological
osteomalacia and has also responded well to oral
phosphate. When X stopped taking oral phosphate,
hypophosphataemia and muscle weakness occurred
within hours. This predictable muscle weakness,
which was rapidly reversed by giving phosphate,
enabled us to study the relationship between
plasma phosphate, muscle phosphate and muscle
function by a variety of methods (Fig. 1).
Four adults (a mother, aged 50 years, and her
three daughters, aged 27, 25 and 23 years) from a
family with the typical features of inherited hypophosphataemia causing rickets from infancy were
also investigated. None was receiving treatment at
the time of investigation and none had subjective
or objective muscle weakness.
Studies were also made on a man aged 34 years
(the offspring of consanguineous parents) who had
the features of tumoral calcinosis with large
calcified masses in the soft tissues, especially over
the right hip, since childhood and persistent hyperphosphataemia without renal failure [ 111.
Methods
During the period of study patient X consumed
a constant diet low in gelatin and 3-MeH. Routine
biochemical methods were used for measurement
of plasma and urine Ca and P, and 3-MeH in the
urine was measured by automatic amino acid
analysis [12]. Muscle strength was monitored as
the force of a maximum isometric voluntary contraction (MVC) of the quadriceps with the knee
flexed to 90" [13]. The mean expected MVC for
the stronger leg in young normal subjects is
330 N and the lower limit (-2 SD) 280 N. The
coefficient of variation of repeated measurements
is 8% [14].
High-resolution 31P 1i.m.r. spectra (Fig. 2) were
recorded with a TMR-32 Fourier transform
spectrometer (Oxford Research Systems) incorporating a 1.89 Tesla superconducting magnet.
The horizontal bore of the instrument was 20 cm
and the distance from the aperture to the sensitive
volume was 35 cm. Consequently the only part of
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Time (days)
FIG. 1. Effect of stopping oral phosphate supplements (P) on plasma phosphate concentration,
urine 3-methylhistidine (3-MeH) excretion, maximum voluntary isometric contraction (MVC) of
the quadriceps, muscle pH and the ratios of
PCr/Pi and PCr/ATP recorded from the forearm
muscles by 31P n.m.r. spectroscopy in patient X
with adult-onset hypophosphataemic osteomalacia.
For normal n.m.r. values see Table 1.
the limb which could be studied was the forearm
and in all cases the spectroscopy was limited to the
belly of the right flexor digitorum superficialis
muscle. A 25 mm diameter surface coil [15] constructed from two turns of 2 mm diameter insulated
copper wire was used for signal detection. Spectra
were recorded at rest at 32.5 MHz using a pulse
width of 20 ps repeated every 1.0 s. Spectra were
also recorded from the same site during an exercise
programme in which the subject squeezed a
sphygmomanometer bulb connected t o a resistance
of 100 mm Hg once every 2 s for 10 min or until
fatigued.
Osteomalacia and n.m.r. spectroscopy
PCr
507
metabolites identified by n.m.r. are proportional
to the intensity of their assigned signals their
relative tissue concentrations could be easily and
accurately computed [ 161.
Unlike the signal of inorganic phosphate the
chemical shift (i.e. the spectral position) of the
phosphocreatine signal is constant throughout the
physiological range of tissue pH. Consequently
intramyocellular pH could be monitored by
measuring the difference in chemical shifts of
these two metabolites and relating the value to a
previously constructed calibration curve [9].
The results of control studies on healthy
volunteers have already been published [ 171.
ATP
P
Results
I
,
5
0
1
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-5
-10
-15
Chemical shift @.p.m.)
-20
FIG. 2. 31P n.m.r. spectrum representing thesum of
256 scans recorded at rest from patient X whilst
taking oral phosphate supplements. The broad
components have been minimized by apodization
and the signal-to-noise ratio improved by line
broadening of 6Hz. Chemical shifts (parts per
million) are defined as positive in the high frequency direction, with the peak of phosphocreatine chosen as the internal chemical shift
standard. Peak assignments are: PCr = phosphocreatine, Pi = inorganic phosphate, Q! = a-phosphate of ATP, ADP plus NAD' and NADH, 0 =
0-phosphate of ATP, y = y-phosphate of ATP
plus /3-phosphate of ADP. The position of the Pi
signal relative to that of PCr defines intramyocellular pH as 7.00.
Absolute quantification of the intramyocellular
phosphate-containing metabolites was not possible
using a surface coil since the exact volume of
tissue that contributed to the signal could not be
determined with accuracy (for other reasons see
[S]). However, since the concentrations of the
In patient X the plasma phosphate concentration
rapidly declined to 0.6 mmol/l when oral phosphate
supplements were discontinued (Fig. 1). This
reduction was associated with a clinical proximal
myopathy and a significant (P< 0.05) reduction
in the MVC of the quadriceps. The excretion of
3-MeH and the ratio of 3-MeH/creatinine (thought
to be an index of the rate of myofibrillar degradation [18,19]) in the urine remained unchanged
[lo3x normal 3-MeH/creatinine excretion is 16*
0.5 (mean f SEM)]. 31P n.m.r. spectra (Fig. 2 )
recorded at rest before phosphate was stopped
revealed a normal intramyocellular pH (normal
range 7.03 t 0.03 SD). The PCr/Pi and PCr/ATP
ratios were both at the lower end of the normal
range (Table 1). Hypophosphataemia produced no
significant change in the n.m.r. spectra recorded at
rest and the changes during exercise did not differ
from the pattern recorded previously in normal
subjects [8]. The findings in patient Y were also
normal.
In the four non-myopathic patients with
inherited hypophosphataemia the recorded n.m.r.
spectra were normal (Table 1). Those recorded from
the patient with inherited hyperphosphataemia
were also normal except for a reduction in the
resting PCr& ratio.
TABLE1. Patients studied and mean n.m.r. values recorded at rest
Condition
Adult-onset
hypophosphataemia
Inherited hypophosphataemia
WDRR)
Idiopathic hyperphosphataemia
Control subjects
*1SD
Patient
X*
Y
(4)
(1)
(20)
Plasma Pi
(mmol/l)
Intracellular
1.20
0.75
0.65
7.00
7.10
1.06
4.9
7.2
10.0
2.1
3.8
2.7
2.20
1.12
k0.16
7.02
7.03
f 0.03
4.8
9.1
* 2.0
2.3
3.2
i0.6
* Values obtained before oral phosphate was stopped.
PCr/Pi
PCr/ATP
PH
508
R. Smith et al.
Discussion
During induced hypophosphataemia in patient X
the constancy of the 3-MeHlcreatinine ratio
probably excluded any marked change in the
myofibrillar breakdown. The observation that the
PCr/Pi and PCr/ATP ratios, although low, remained
unchanged was compatible with the absence of
any gross change in muscle phosphate metabolism
during hypophosphataemia, but did not exclude
changes in the absolute concentrations of the
measured phosphorus-containing metabolites. Previous studies in vitro [ 171 and in vivo on exercising
human 18,201,ischaemic rat [21,22] and ischaemic
human [23] muscle as well as investigations on
pathological degenerating rat muscle [ 71 have
shown that ATP levels remain normal until PCr
is depleted. Thus the finding in this hy?ophosphataemic patient of an unchanged ratio of PCr
to ATP implies that the absolute level of PCr does
not alter even in the presence of muscle weakness.
The reason why both resting PCr/ATP and PCr/Pi
ratios were at the lower limit of the normal range
in this patient is unknown. It is possible that the
apparent constancy of the n.m.r. data recorded
from the distal muscles at the time of documented
proximal myopathy is accounted for by biochemical differences between proximal and distal muscle
groups. The relatively small bore of n.m.r. spectrometers currently available precludes the study of
the proximal girdle musculature.
In patients with inherited hypophosphataemia
phosphate transport is abnormal in renal tubular
cells [24] and the concentration of Pi within
circulating erythrocytes, leucocytes and platelets
has been reported to be half normal whilst that of
ATP is within the normal range [25]. In this disorder
the level of Pi within the muscle cells is unknown
but these n.m.r. studies raise the interesting
possibility that the intramyocellular concentration
of Pi is in fact normal. This would increase the
resting gradient for Pi across the cell membrane, a
process which would require increased energy
expenditure.
Hyperphosphataemic tumoral calcinosis is an
inherited condition which is the biochemical
‘opposite’ of inherited hypophosphataemia with an
increase, rather than a decrease, in the maximum
renal tubular reabsorption of phosphate (Tm,pi/
GFR). A possible explanation for the low PCr/Pi
ratio found in the single patient studied is that the
intracellular Pi was increased (the contribution to
the spectra from the extracellular Pi is negligible
[8l>.
These preliminary, non-invasive studies in vivo
in patients with abnormal plasma phosphate levels
suggests that the myocellular and extracellular
phosphate concentration are not directly related.
Further investigation is needed to determine why
myopathy occurs in some hypophosphataemic
states and not in others.
Acknowledgments
We thank the British Heart Foundation and the
Medical Research Council for financial support.
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