36p
Medical Research Society
There was also no significant difference in frequencies
between those with and without clinically detectable CHD
or between those with and without a history of CHD before
age 60 in a first degree relative. CHD patients had the
same allele frequencies as those in the lowest fifth of a
CHD risk score (Shaper et aI, BMJ, 1986, 293, 474-479).
Haplotype analysis identified 11 haplotypes in 32 genotypes. The distribution of the genotypes in the 661 men
and in all but one of the subgroups did not depart
significantly from Hardy-Weinberg predictions. The
exception was a subgroup with a positive paternal history
of CHD, %2(65df)=128.2; P(O.OOI. The high value of %2
could be accounted for by the genotypes of 2 men who were
heterozygous at the Xmn I, Msp I and Pst 1 sites. Neither
was hyperlipidaemic or had clinically detectable CHD.
greater than 8mg/l (the detection limit of the assay)
with a maximum concentration of Il6mg/l. Plasma Zn
concentration was 12.2±2.2umol/l (range: 7.7 - 17.8,
normal range 11 - 18). There was no correlation between
Zn and IGF-l, but there was a significant correlation
between CRP and IGF-l (r =-0.52; p <0.02).
These results suggest a possible role for the mediators
of the acute phase response in the regulation of IGF-l
synthesis and secretion in chronic renal failure.
137 WHOLE BODY LEUCINE KINETICS AND FRACTIONAL QUADRICEPS MUSCLE SYNTHETIC RATE (MPSR) IN ALCOHOLIC PATIENTS
P.J.PACY, M.READ, V.PREEDY*, T.J.PETERS* & D.HALLIDAY
Nutrition Research Group and *Division of Clinical Cell
Biology, MRC Clinical Research Centre, Harrow HAl 3UJ.
LEUCOCYTE COPROPORPHYRINOGEN III OXIDASE:
DEVELOPMENT AND CLINICAL APPLICATIONS OF A SENSITIVE AND
SPECIFIC HPLC ASSAY.
135
Rong Guo, C K Lim, K J Simpson and T J Peters. Division
of Clinical Cell Biology, MRC Clinical Research Centre,
Watford Road, Harrow, Middlesex HA1 3UJ. UK.
An accurate, sensitive and specific HPLC assay is
described for coproporphyrinogen oxidase (EC 1.3.3.3)in
human peripheral blood leucocytes. The standard
incubation mixture was 100~1 enzyme solution (approx. 1mg
protein), and 100~1 0.25M Tris/HCL buffer containing 1mM
EDTA, pH 7.0, preincubated at 37 0C for 5min. The reaction
was started by adding 50~1 of freshly prepared
coproporphyrinogen III (final concentration 1~M) in Tris/
HCl buffer containing 0.1M ascorbic acid and 1mM EDTA, pH
7.0, and incubation was continued for 1h at 37 0C in the
dark. The reaction was terminated by vortex-mixing with
250~1 of ice-cold 20% (W/V) Trichloroacetic acid/DMSO
(1:1,v/v) containing 42nM mesoporphyrin as an internal
standard. After centrifugation, 200~1 of the supernatant
was analysed by HPLC on a 25cm x 5mm ODS-hypersil column
with 88% (v/v) methanol in 1M ammonium acetate bUffer,
pH 5.16 as eluent. The flow rate was 1.5ml/min. A
fluorometer was used for detection at excitation 400nm
and emission 618nm. Protoporphyrin IX formation
increased linearly up to 1h and up to 2mg leucocyte
protein. The Km was 0.12 ± 0.021~. Dithiothreitol,
glutathione and cysteine all inhibited more than 30% of
the activity. The mean activity for 43 normal subjects
was 0.239nmol/h/mg protein and the reference range was
established as 0.101--0.377 (mean ± 2SD). In patients
with hereditary coproporphyria, the mean activity (0.0747
± 0.0334; n=4) was significantly lower than the reference
range. In alcoholic patients, the mean activity (0.316 ±
0.115; n=24) was approx. 30% higher than normal controls.
This method is thus useful in the differential diagnosis
of the porphyrias.
Proximal myopathy is a well established clinical feature
of alcohol abuse but no data are available on muscle
protein synthesis rate (MPSR). This is determined by
documenting the rate of incorporation of l3C-leucine into
muscle during a primed constant infusion of L_(1_13C)
leucine (1). The aim of the present study was to determine post-absorptive whole body leucine kinetics using
the reciprocal (2) pool model and fractional MPSR (%h-I )
in established alcoholic patients. Their clinical details
were M4, F2; age 47±I2 years, weight 69±7 kg. We have
compared data with historical controls (M18, age 29±6
years; weight 72±6 kg). The protocol consisted of an 8
hour L-(I-I3c)leucine infusion (Img kg-Ih- I) with quadriceps biopsies (200mg) obtained at two and eight hours
i.e. during plasma plateau enrichment of 13C KIC which
closely approximates that of intracellular muscle 13C_
leucine enrichment (3). Plasma 13c aKIC enrichment was
determined by GC-MS while 13C02 in expired breath and
13C-leucine in muscle was by IRMS. The results (mean±SD)
are shown below:
Alcoholics
Controls
MPSR
Whole body leucine kinetics
(%h- I)
(~mol kg-Ih- I)
Oxidation Synthesis
Flux
0.027±0.009
14±4
83±9
97±IO
15±4
10I±10** 0.046±0.008**
116±I2**
Statistics by unpaired t-test
**p<O.OOl
Results indicate that both whole body leucine flux and
protein synthesis and fractional MPSR are lower in
alcoholic than corresponding values obtained in control
subjects.
1) Halliday D. et aI, Clin Sci 1988; 74: 237-240
2) Layman D.K. et aI, Am J Physiol 1987; 253: EI73-EI78
3) Schwenk W.F. et aI, Am J Physiol 1985; 249: E646-E650
POST-ABSORPTIVE WHOLE BODY LEUCINE KINETICS AND
QUADRICEPS MUSCLE PROTEIN SYNTHETIC RATE (MPSR) IN THE
POST-VIRAL SYNDROME
138
P.J. PACY, M. READ, T.J. PETERS* AND D. HALLIDAY
Nutrition Research Group & *Division of Clinical Cell
Biology, MRC Clinical Research Centre, Harrow HAl 3UJ
INFLUENCE OF THE ACUTE PHASE RESPONSE ON CIRCULATING
INSULIN-LIKE GROWTH FACTOR 1 (IGF-l) IN HAEMODIALYSIS
PATIENTS.
136
As the name implies this syndrome is associated with
severe lethargy and listlessness following a viral illness. This has long been recognised but the paucity of
documented abnormalities has raised the possibility of a
psychological rather than physical aetiology. A predomin-
B Sampson. G Carter, J Alaghband-Zadeh and JR Curtis.
ant feature in post-viral individuals is severe muscle
Departments: Chemical Pathology and Medicine,
Charing Cross Hospital, Fulham Palace Road, London, W6 8RF
uced muscle RNA content was suggestive of reduced muscle
Serum IGF-l concentrations are known to be low in
haemodialysis (HD) patients, with raised serum growth
hormone (GH). These patients are also known to have
raised serum C-reactive protein (CRP) and reduced plasma
zinc. In order to study the relationship between
circulating IGF-l levels, the acute phase response and
zinc. serum IGF-l, GH, CRP and plasma zinc were measured
in 21 HD patients.
+
The mean serum IGF-l concentration found was 0.28-0.23u/ml
(range: <0.1 - 0.96; normal range: 0.6 - 1.8). GH
concentration was 16.2±17.1mu/l (range: 1.8 - 81.2;
normal range: <10). 17 patients had a CRP concentration
weakness, but not wasting, although the finding of redprotein synthesis. The aim of this study was to evaluate
whole body leucine kinetics and fractional MPSR by stable
isotope techniques (1) in five post-absorptive females
with the syndrome. Their clinical details were: age 38±2
years; weight 56±8 kg; body mass index (kg/m 2) 21±3.
Whole body leucine kinetics were determined by primed
continuous infusion of L-(1-I3C) leucine (lmg kg-Ih-I)
for eight hours. Two muscle biopsies (200mg) were removed
six hours apart during isotopic plateau enrichment of
plasma 13C KIC which was taken to represent precursor
pool labelling (2). It is assumed that tracer incorporation into muscle was linear between biopsies. Subsequent
data was compared with that from controls (18M: age 29±6
years; weight 72±6 kg; BMI 23±2). In contrast with
..,
37
Medical Research Society
controls post-viral syndrome patients had significantly
reduced whole body leucine flux (116±12 versus 83±7;
p<O.OOl) and protein synthesis (101±10 versus 69±8;
p<O.OOl) while whole body leucine oxidation was comparable. (Figures are mean±SD and units ~mol kg-lh- l.)
Fractional MPSR (%h- l) was lower (0.046±a.008 versus
0.034±0.009 p<0.05). We tentatively suggest that the postviral syndrome is associated with reduced protein
synthesis in skeletal muscle.
(1) Halliday D. et al. Clin Sci 1988; 74: 237-240.
(2) Layman D.K. et al. Am J Physiol 1987; 253: E173-E178
139 MEASUREMENT OF WHOLE BODY PROTEIN TURNOVER IN MAN
USING A (2H5)PHENYLALANINE MODEL
GN THOMPSON, PJ PACY, GC FORD, H MERRITT, MA READ,
AJ NOBLE AND D HALLIDAY
Nutrition Research Group, Clinical Research Centre,
Harrow HAl 3UJ, England.
A method of measuring whole body protein turnover based
on primed constant infusions of (2 H5)phenylalanine (0.5rng!
kg/h) and (2H2)tyrosine (0.25mg/kg/h) has been validated
against the widely employed (13C)leucine method in 6
normal adults and in 2 children with Lesch-Nyhan syndrome.
To hasten achievement of isotopic plateaus, priming doses
of (2 H5)phenylalanine (0.5mg/kg), (2H2)tyrosine (0.25mg/
kg) and (2H4)tyrosine (0.08mg/kg) were given. Phenylalanine and tyrosine concentration (using a-methylphenylalanine and a-methyl tyrosine as internal standards) and
enrichment were measured in plasma by GCMS using previously described GCMS methods (1). Plasma a-ketoisocaproic acid (KIC) enrichment was used to calculate leucine
turnover; (2H5)phenylalanine and (2H2)tyrosine enrichments were corrected to mixed venous values (2). Phenylalanine hydroxylation and flux were calculated as described by Clarke and Bier (3) who demonstrated attainment
of tyrosine enrichment plateau after 6-8 hours. By using
the priming doses described above, enrichments of all
measured isotopic species reached plateau within 2 hours
of constant infusion. In adults the phenylalanine
method (mean±SD protein synthesis 3.00±0.15 g/kg/d,
catabolism 4.04±0.29) gave similar results to the leucine
method (synthesis 3.09±0.27, catabolism 3.70±0.35), and
both methods again gave similar values in each child. The
phenylalanine method appears to reflect protein metabolism in a similar manner to the leucine method. It is
comparable also in the short study period required, but
has the additional advantage of not requiring measurement
of expired CO 2 production rate or enrichment.
1) Schwenk WF et al. Anal Biochem 1984; 141: 101
2) Layman DK, Wolfe RR. Am J Physiol 1987; 253: E173
3) Clarke JTR, Bier DM. Metabolism 1982; 31: 999
140 Body cell mass following major electice surgery.
F. Carli and C. Freemantle (Introduced by Dr. J. Milledge)
Department of Anaesthesia and MRC Cyclotron Unit. Post
graduate Medical School, Hammersmith Hospital, London W12.
Surgery in normal SUbjects results in a short catabolic
period which is characterised by weight loss, negative
nitrogen balance with positive salt and water balance
suggesting a post-operative decrease in body cell mass.
The size of body cell mass can be estimated by measuring
total body potassium (TBK). Potassium is the most abundant
intracellular cation which is linearly related to the Slze
of the body cell mass 1• Limited data is available on :BK
during the post-operative period. 66 patients undergolng
either intra-abdominal, pelvic, orthopaedlc
cardla~
surgery were studied. Age,sex,body weight,helght, sklnfold
thickness were recorded before surgery. TBK wa~ measured
noninvasively by use of a whole body counter wlth a
0:
p
pr-ec ts Ionof' + 2't,. The measurements occurred before surgery,
and four and-seven days after surgery. The results of the
study are summarised in the table below.
Before Surgery
Mean + (1SD)
Abdominal
2811 (+142)
n=10
2661 (+404)
Pelvic
n=18
Orthopaedic
2582 (+685)
n=23
Cardiac
3321 (+185)
n=15
~**p
*p
0.05 **p 0.01
After Surgery
4 days
1 days
*
2510 (+668)
**
2654 ~+152)
**
2584 (+412)
*
2654 (+319)
***
2321 (+511 )
***
2312 (+555)
3113 (+149)
0.001
There was a significant drop in TBK in all groups
following surgery and the greatest fall occurred at day 4.
No correlation was found between the percentage of
measured over predicted and the post-operative TBK loss.
TBK fell by 3% after pelvic surgery compared to other
groups (1%) suggesting that the decrease in body cell mass
might be related to the severity of surgical trauma.
1. FD Moore et al. The body cell mass and its supporting
enviroment. Philadelphia 1963, WB Saunders.
THE ROLE OF ATRIAL NATRIUBETIC PEP1'IDE
IN THE NATRIURESIS OF BEAD-OOWN TILT
MJ ALLEN, VTY ANG and ED BENNETT
St George's Hospital Medical School, London
Head-down tilt results in atrial stretch, the release of atrial
natriuretic peptides (ANP) and a natriuresis, but a direct
cause and effect relationship has not been fully established.
Eight volunteers took 500mg lithium carbonate the evening
before each study and attended on 3 occasions. After a 60
min control period sitting upright they underwent a 3-hr
infusion of ANP (1.2 pmol/kg/min), a 3-hr placebo infusion
and a 3-hr period oflOohead-down tilt, in random order.
Fractional excretion of sodium was unchanged with
placebo, increased with ANP from 0.83 ± 0.16 to 1.12 ± 0.12
(mean ± SEM, P<O.Ol) and increased with tilt from 0.87 ±
0.12 to 1.34:t 0.10 (P<O.OOl). The urinary volume response to
ANP (5.8 ± 1.2 rising to 7.9 ± 0.5 ml/min) did not differ from
placebo. Tilt produced a diuresis (5.6 ± 1.2 rising to 10.8 ±
0.4 ml/min; P<O.OOl) which was significantly greater than
placebo or ANP (P<O.OOl) [probably reflecting additional
inhibition of vasopressin]. Lithium clearance (equivalent to
sodium clearance by the proximal nephron) increased with
both tilt and ANP. A delayed increase in sodium clearance
by the distal nephron was more marked with tilt than ANP
[perhaps due to changes in either aldosterone or a
hypothalamic NaIK ATP-ase inhibitor]. Haematocrit was
unchanged by placebo, showed a modest increase with
ANP (43.5 ± 0.9 to 44.3 ± 0.6: P<0.05), and a modest
reduction with head-down tilt (43.8 ± 0.9 to 43.1 ± 0.9: ns),
Plasma ANP was unchanged by placebo, rose with tilt from
8.1 ± 1.0 to 11.4 ± 2.5 (P<O.OOl) and with ANP infusion from
6.5 ± 1.4 to 32.3 ± 10.7 pg/ml (P<O.OOl). Plasma ANP was
significantly greater following ANP infusion than during
tilt even though natriuretic responses were similar. These
results suggest that ANP alone is a less potent natriuretic
than head-down tilt. This may be because ANP is not the
major determinant of the natriuretic response to tilt or that
the effects of ANP are dependent on other responses to tilt
such as a reduction in renal sympathetic nerve activity or
reduced activity in the renin-angiotensin-aldosterone axis.
141
142 EFFECT OF ABNOR~~ ~lliGATIVE INTRATHORACIC PRESSURE
ON ATRIAL NATRIURETIC PEPTIDE (ANP) R~'LEASE AND RENAL
FUNCTION IN HEALTHY MEN.
A WARLEY, F FONTES. M WILSON. A RAINE. J S~LING.
Osler Chest Unit and Nuffield Dept of Med. Oxford.
In health. inspiration is normally accomplished.by
enerating -5 cmsH20 intrapleural pressure (relat~ve to
~tmospheric). When upper (eg obstructive sleep apnoea -