Medical Research Society
2 4 ~
Ten healthy subjects (five females, five males, age 24 f 4
years) were fasted overnight. Each received soluble insulin (0.15
unit/kg) intravenously, and blood was withdrawn from an indwelling cannula into chilled heparin-treated tubes at intervals of
15-30 min for 2 h. Blood pressure was recorded at intervals of 2
min throughout the study. Plasma was separated at 4OC within
30 min of drawing blood to measure plasma glucose (pG),
plasma arginine vasopressin (pVP), plasma osmolality, plasma
sodium (pNa) and potassium (pK) and packed cell volume. One
subject was water-loaded (20 ml/kg) before repeating the study.
Urine was collected before and during the study to measure
urine osmolality.
The fall in pG from 4.7 f 0 . 2 (mean f SEM)to 1.6 f 0.1
mmol/kg was associated with a rise in pVP from 1.2 ?r 3 to 8.2
k 3 . 6 pg/ml. P < 0.01, which occurred at 30 min. There was no
significant change in plasma osmolality, but pNa rose slightly at
30 min, basal 138.4 f 0.5, peak 140.4 f 0.5 mmol/l, P < 0.01,
and pK fell by I .O mmol/l after 45 min. Packed cell volume did
not change significantly indicating no large change in plasma
volume. Furthermore, there was no significant change in mean
arterial blood pressure; pulse rate rose from 6 1 . 2 ?r 2 . 3 to 94.3
k 7.1 beadmin. The subject who was water-loaded before
repeating the study showed a rise in pVP that was associated
with an inability to excrete the water load (45% excreted at 2 . 5
h, normal > 80%) and rise in urine osmolality from 84 to 441
mmollkg.
We conclude that the rise in pVP in response to hypoglycaemia was not mediated by changes in osmolality, blood
pressure or volume, but by an independent mechanism.
80. EFFECT O F PREGANGLIONIC SYMPATHECTOMY
ON
METABOLIC
RECOVERY
FROM
HYPOGLYCAEMIA
79. ADENOSINE DIPHOSPHATE-INDUCED PLATELET
SHAPE-CHANGE IN NORMAL AND DIABETIC
SUBJECTS
81. A METHOD FOR THE PRE- AND POST-NATAL
OF DEFECTS O F ISOVALERATE
DETECTION
METABOLISM
-
B. M. FRIEQ R. J. M. CORRALL,J. P. ASHBY,E. J.
MCCLEMONT
AND P. s. SEVER
W.
Metabolic Unit, University Department of Medicine, Western
General Hospital, Edinburgh, Edenhall Hospital, Musselburgh.
East Lothian, Scotland, U.K.. and Medical Unit. St Mary's
Hospital, London
To evaluate the role of the sympathetic nervous system in the
recovery from acute hypoglycaemia, six male subjects with
complete post-traumatic transection of the cervical spinal cord
were studied, and the results compared with I 1 normal controls.
Insulin (0.I 5 unit/kg) was administered intravenously to fasting
subjects and sequential blood samples were assayed. The
recovery of blood glucose after hypoglycaemia was unimpaired
in the sympathectomized subjects, although a rise in blood
lactate concentration was not observed in this group. Changes in
plasma free fatty acid and glucagon concentrations were similar
in both groups. The marked increase in plasma cyclic-AMP
and plasma noradrenaline concentrations seen in normal
subjects was absent in the sympathectornized group.
This study demonstrates that, after a total preganglionic sympathectomy in man, blood glucose recovery from insulininduced hypoglycaemia is normal. Glucagon secretion appears
to be intact, but a rise in plasma cyclic-AMP concentrations
does not occur. The putative role of adrenergic mechanisms in
the homeostatic recovery from hypoglycaemia in man may
require re-evaluation.
AND EVAKOHNER
MASSIMO
PORTA', P. HILCARD
R. A. CHALMERS
AND E. SPELLACY
Endocrine Unit and 'Haematology Department, Hammersmith
Hospital, London
Dioision of Inherited Metabolic Diseases, MRC Clinical
Research Centre, Harrow, Middlesex. U.K.
Platelet hypersensitivity to aggregating agents in oitro has been
reported by many authors in diabetic patients, and has been
suggested to be of importance in the pathogenesis of diabetic
microangiopathy. As part of a detailed study on blood coagulation in diabetes, we have studied platelet function in 64
subjects (29 control and 35 diabetic patients). Besides the
conventional aggregation studies, we also studied induction of
shape-change in calcium-deprived platelet-rich plasma.
Conventional aggregrometry studies revealed that patients
with severe retinopathy had a lower threshold for ADP-induced
aggregation than did those with milder forms of the disease and
controls. No difference between diabetic patients and controls
was found by other aggregation techniques; collagen ( I mg/ml),
ristocetin (2 mg/ml) and ADP (5 pg/ml). In particular, patients
with pre-proliferative lesions were not shown to be different from
normal subjects.
ADP (5 pg/ml)-induced shape-change of platelets was
recorded in the presence of EDTA (7.6 mmol). The tracings of
the controls were measured and compared with those of patients
with diabetic retinopathy. Significant differences (P < 0401)
were found between controls (12.58 2 SEM 0.68%) and diabetic
subjects with proliferative retinopathy (17.25 f 1.07%).
Patients with background retinopathy (13.97 f 1.48%) and
maculopathy (14.89 k 2.01%) did not differ significantly from
normal subjects.
Shape-change in platelets is the first step leading to platelet
aggregation. These observations therefore indicate that abnormalities in platelets from diabetic patients may be detected in the
initial steps of aggregation, and this could be reflected in an
increased activity in oiuo.
Methodological studies showed that this test is reproducible
and simple, and may therefore be useful in the evaluation of
platelet function in diabetic patients.
At least five clinically important inborn errors of metabolism are
known, due to specific deficiencies of the four enzymes and
associated cofactors involved in the metabolism of isovalerylCoA to acetyl-CoA in the leucine metabolic pathway. We report
here a simple rapid method for their detection in a single assay
that requires relatively few cultured cells and is suitable for both
pre- and post-natal detection of these diseases.
Skin fibroblasts, or amniotic cells obtained by amniocentesis
at 16-18 weeks' gestation, are grown to confluence in Eagle's
minimum essential medium (biotin-free) supplemented with 10%
foetal calf serum and with added penicillin and streptomycin. At
confluency, the cells are harvested by trypsinization, and seeded
into 25 cm2 Falcon assay flasks. After continued culture to 5090% confluence, the monolayers are rinsed, and incubated for
18 h at 37OC in Puck's solution A with glucose and 15% foetal
calf serum, with added [ I-14Clisovalerate.Control assays with
II-"Clacetyl-CoA are also made. The Puck's solution is then
removed, the cells are rinsed, and the cell protein is precipitated
with trichloroacetic acid. After further rinsing and drying, the
protein is dissolved and the "C radioactivity and total protein
content are determined.
Fibroblasts from control subjects showed an incorporation of
isovalerate into protein of 196 f 4 1 pmol h-' mg-I of protein,
and control amniotic cells an incorporation of 181 2 51 prnol
h - I mg-' of protein. Fibroblasts from a patient with proven isovaleric acidaemia showed an incorporation of 13 ? 3 pmol h-I
mg-I of protein (6.6% of control fibroblast values). The assay
has also been applied to the prenatal diagnosis of 3-methylcrotonylglycinuria, in which an unaffected foetus has been predicted [incorporation of isovalerate into protein of 103 pmol h-I
mg-I of protein (57% of control amniotic cell lines)l. Incorporation of II-"CIacetyl-CoA into protein was similar in all cell
lines studied (average 126 pmol h-I mg-I of protein).