186

TIPS - May 1986

Hypoglycin, the famous toxin of

the unripe Jamaicanackee fruit
H. S. A. Sherratt
Unripe fruit of the Jamaican ackee tree, Blighia sapida, contains hypoglycin,
L-(methylenecyclopropyl)alanine), a hypoglycaemic toxin which has caused an
estimated 5000 deaths. Methylenecyclopropylacetyl-CoA, a metabolite of
hypoglycin, inactivates several, but not all, flavoprotein acyl-CoA dehydrogenases causing widespread disturbances of the oxidation of fatty acids and
several amino acids leading to secondary inhibition of gluconeogenesis. Despite
intensive study for three decades the mechanism of hypoglycin poisoning is still
only partly understood. H. S. A. Sherratt reviews these complex effects which
have implications for toxicology and the study of metabolic regulation and of
many inborn errors of metabolism.
The ackee tree was imported from
West Africa to Jamaica where it
was named Blighia sapida, this
introduction being wrongly attributed to Captain Bligh of Bounty
fame. Its fruit are about the size
of an orange with a thick rind
containing
seeds
resembling
chestnuts (Fig. 1). The seeds are
surrounded by a fleshy arillus
which is a regular feature of the
Jamaican diet. Eating unripe ackee
fruit often causes vomiting sickness with the onset of symptoms
about 2-3 h later. Severe hypoglycaemia and vomiting, depletion
of liver glycogen, accumulation of
fat in the liver, increased plasma
free fatty acid concentrations and
death often occur. More recently,
massive dicarboxylic aciduria and
some organic acidaemia has been
observed 1. Between 1886 and 1950
vomiting sickness in Jamaica caused approximately 5000 deaths 2.
Although the toxic nature of unripe
arrilli is now recognized in Jamaica
there are still occasional cases of
poisoning.
Two toxic compounds, hypoglycins A and B, were isolated from
ackee seeds by Hassal and Reyle in
1954. Hypoglycin (hypoglycin A)
is an unusual amino acid, L- (methylenecyclopropyl)alanine, and hypoglycin B is 7-glutamylhypoglycin
H. S. A. Sherratt is a reader in biochemical
pharmacology at the Departmentof Pharmacological Sciences, University of Newcastle
upon Tyne, Newcastleupon Tyne NE2 4HH,
UK.
1986, Elsevier Science P u b l i s h e ~ B.V., A m s t e r d a m

0165 -

(Fig. 2). Hypoglycin (either given

orally or parenterally) causes hypoglycaemia in fasted animals in
doses ranging from 10-150 mg kg -1
body weight in guinea-pigs, rabbits, dogs, cats, rats and mice with
decreasing sensitivity. The toxic
dose in man is unknown but may
be much less on a per/kg basis.
Behavioural depression, and, in

small animals, hypothermia with

body temperatures as low as 25C
(when the ambient temperature is
20C), are features of hypoglycin
poisoning.
Hypoglycin is not readily available and has not been synthesized
in useful quantities. It also occurs
in a few other plants of the Acer
family, including seeds of the
common sycamore Acer pseudoplatinatus. Since its isolation there
has been a sustained interest in
hypoglycin, and this review will
mainly describe work during the
last 15 years. Space does not allow
mention of all workers who have
contributed and further details are
given elsewhere (Refs 3-7).
Primary metabolic effects:
inhibition of fatty acid oxidation
Hypoglycin causes profound
disturbances of the oxidation of
fatty acids, some amino acids, and
inhibition of gluconeogenesis.
Pioneering work by yon Holt
established that hypoglycin is
converted in vivo to methylenecyclopropylpyruvate (MCPP) which
is then oxidatively decarboxylated
to
methylenecyclopropylacetate
(MCPA) as its CoA ester (MCPACoA). MCPA-CoA may be reversibly converted to free MCPA or

Fig. 1. Ripe ackee fruit, Blighia sapida. The arillus surrounding the seeds is cooked and
eaten, for example as 'ackee and salt fish; When unripe the ari/li contain dangerous
amounts of hypoglycin and hypoglycin B. During ripening these compounds are
translocated into the seeds.
6147186/$02.00

187

TIPS - M a y 1986

conjugated with glycine to MCPAglycine (Fig. 3). The metabolic

disturbances are consequences of
the inactivation by MCPA-CoA in
the mitochondrial matrix of the
general acyl-CoA and butyryl-CoA
dehydrogenases involved in ~oxidation, and the isovaleryl-CoA,
2-methylbutyryl-CoA and glutarylCoA dehydrogenases involved in
amino acid catabolism 8-1. MCPACoA is a suicide inhibitor of the
general-acyl-CoA
dehydrogenase m (and presumably of the other
enzymes). An early step in the 2,3dehydrogenation of acyl-CoA esters catalysed by this enzyme is
removal of a proton from the 2position. MCPA-CoA gives an
active species which combines
with the flavinadenine dinucleotide prosthetic groups of the enzyme to form modified flavin
derivatives 1. In addition, mitochondrial NADPH-dependent 4enoyl-CoA reductase which is not a
flavoprotein may be inhibited.
Hypoglycin and MCPP have other,
apparently less important effects.
It was shown in 1958 that several
carboxylic acids with the structure

CH2=C.C.C.COOH are hypoglycaemic (Fig. 2). Of these, pent-4enoate has been extensively investigated because it was once
believed to be an analogue of
MCPA. However its mechanism of
action differs from that of hypoglycin (see Refs 6, 7).
The complete mitochondrial oxidation of long-chain fatty acids
requires the successive action of
palmitoyl-CoA, general acyl-CoA
and butyryl-CoA dehydrogenases
as the carbon chain is shortened by
successive loss of acetyl units.
MCPA-CoA inactivates the latter
two enzymes so that long-chain
acyl-CoA esters are only oxidized
as far as butyryl-CoA. ButyrylCoA accumulated is then converted to butyrate and CoASH by a
high Km acyl-CoA hydrolase. This
partial oxidation by inhibited liver
mitochondria is clearly shown
with conditions where the acetyl
groups formed by ~-oxidation are
quantitatively converted to acetoacetate 9 (Fig. 4). The maximum
possible rate of generation of
acetyl units from fatty acids is also
decreased by about half. This

hypoglycin (L-(methylenecyclopropyl)alanine)

hypoglycin B (%'-glutamylhypoglycin)

CHz~C

\*

CH.CH2.CH(NH2).COOH

CH2
~H.CH2.CH.COOH
I
NH.CO.CH2.CH2.CH(NH2).COOH
CHa

methylenecyclopropylpyruvate (MCPP)

CH2=C /

methylenecyclopropylacetate (MCPA)

Dicarboxylic aciduria
Hypoglycin-poisoned rats excrete large amounts of glutaric
acid 1. Glutaryl-CoA is an intermediate in the degradation of
tryptophan and lysine. Inhibition
of glutaryl-CoA dehydrogenase by
MCPA-CoA, followed by deacylation of accumulated glutaryl-CoA
yields glutarate. Rats also excrete

CH2

CH2=C

limited rate is largely set by the

rate of recycling of CoASH (necessary for other stages of ~-oxidation) and by the activity
of palmitoyl-CoA dehydrogenase.
Following administration of hypoglycaemic doses of hypoglycin to
rats, inhibition of butyryl-CoA
dehydrogenase is virtually complete in liver and fatty acid
oxidation is impaired in other
tissues 6. Although acyl-CoA/
CoASH ratios are increased in
liver, 40-50% of the total CoA
remains in the free form (CoASH)
(P. P. Koundakjian, unpublished
results). Free butyrate is found in
plasma during hypoglycin poisoning (up to 0.7 mM in rats and 3 mM
in micen).

CH.CH
*
2. C O . C O 2

CHz
CH2=C/

~ ~H.CH..CO~-

H2-~H.CO23-methylenecyclobutanecarboxylate
CHz=CH--CH 2

pent-4-enoate

CH2=CH.CH~.CH2.CO~-

Fig. 2. Hypoglycin and some related compounds. Hypoglycin is an L-amino acid, C~is asymmetric, and the structure is thought to be (+)(2S: 4S)-2-amino-4,5-methanohexo5-enoicacid. MCPP and MCPA (as its CoA-estor) are derived from hypoglycin in vivo (see Fig. 3). Most
preparations of hypoglycin contain up to 20% of leucine, pure hypoglycin is obtained by hydrolysis of hypoglycin B first separated from
leucine by ion-exchange chromatography. 3-Methylenecyciobutanecarboxylate is a synthetic analogue of hypoglycin. All these compounds
contain the grouping CH2 = CH C.C.COOH. MCPA-CoA (and possibly 3-methylenecyciobutanecarbonyI-CoA) appears to undergo
2-deprotonization by some acyI-CoA dehydrogenases to form suicide inhibitors. Pent-4-enoyI-CoA formed in vivo is a substrato for butyrylCoA dehydrogenase without inhibiting it, and is partly metabolized to 3-oxopent-4-enoyI-CoA which inhibits mitochondrial ~-oxidation by
inactivating the 3-oxoacyI-CoA thiolases.

188

TIPS - May 1986

hypoglycin _

2-oxoglutarate

glutamate ~'-

-- MCPP

NAD

~NADH+H+/

MCPA-CoA

MCPA

AMP + PPi ~

"

\ CoASH + ATP

-glycine

CoASH

MCPA-glycine
Fig. 3. Metabolism of hypoglycin. MCPA-CoA is formed in mitochond/~a by the enzymes which catalyse the degradation of branched-chain
amino acids. MCPA-CoA maybe hydrolysed to free MCPA which is partly reconverted to MCPA-CoA, or partly conjugated with glycine. The
enzymes catalysing these reactions are: I. Leucine-2-oxoglutarate aminotransferase in the cell cytosol 2. Branched-chain 2-oxoacid
dehydrogenase in the mitochondrial matrix 3. Glycine N-acylase in the matrix 4. Medium-chain acyI-CoA hydrolase in the matrix 5. ButyrylCoA synthase in the mattYx.

several other m e d i u m - c h a i n dicarboxylic acids including some

saturated sebacic (C10) and suberic
(C8) acids, and larger amounts of
unsaturated acids particularly 4cis-decene-l,lO-dioic and 4-cisoctene-l,8-dioic acids. Suberic and
sebacic acids are formed w h e n
hepatic mitochondrial B-oxidation
is overloaded in diabetic ketoacidosis, or is impaired in some
inborn errors of metabolism such

as general acyl-CoA-dehydrogenase deficiency, or after administration of drugs which i n h i b i t

mitochondrial B-oxidation (see
Ref. 12). W i t h such conditions
some excess long-chain fatty acids
supplied to the liver are converted
to long-chain dicarboxylic acids b y
c0-oxidation by mixed function
oxidases in the endoplasmic reticulum. Mono-CoA esters of these
dicarboxylic acids then formed

The relation between gluconeogenesis

and fatty acid oxidation
glucose

L
I

glycemld.ehyi.3"phosphate
NAD +

1,3-diphosphoglycerate

phosph~nop~mvate
cytoso]

outside the mitochondrial matrix

are substrates for partial B-oxidation in the peroxisomes where
they are chain-shortened to m e d ium-chain
mono-CoA
esters.
These are then deacylated to nontoxic m e d i u m - c h a i n dicarboxylic
acids.
B-Oxidation in peroxisomes differs from that in mitochondria and
the 2,3-dehydrogenation steps are
catalysed b y the flavoprotein acylCoA oxidase with the concomitant
formation of H202. Acyl-CoA oxidase does not appear to be
inhibited, although it is not k n o w n
whether this enzyme is resistant to
inhibition, or whether any MCPACoA occurs outside the mitochondrial matrix. Experiments
with 14C-labelled fatty acids have
shown that 4-cis-decene-l,lO-dioic
and 4-cis-octene-l,8-dioic acids
are derived from the polyunsaturated fatty acid, linolenate, b u t not
from stearate or oleate 13. Their
formation m a y be explained as a
consequence of i n h i b i t i o n of an
auxiliary enzyme of B-oxidation
necessary for the degradation of
polyunsaturated fatty acids with
double b o n d s in the 9,10 and 12,13
positions, p r o b a b l y the NADPHd e p e n d e n t 4-enoyl-CoA reduc-

tase 13.

J3-oxidation J

rm'toch ondn'a/raatr/x

I n h i b i t i o n of branched-chain fatty
acid o x i d a t i o n

The
branched-chain
amino
acids leucine, isoleucine and valine are transaminated to the corr e s p o n d i n g 2-oxoacids which are
then oxidatively decarboxylated to
isovaleryl-CoA, 2-methylbutyryl-

189

TIPS - M a y 1986

CoA and isobutyryl-CoA respectively. Their further catabolism

involves 2,3-dehydrogenation by
the specific isovaleryl-CoA or 2methylbutyryl-CoA dehydrogenases. MCPA-CoA inactivates these
dehydrogenases and some of the
branched-chain CoA-esters which
accumulate are deacylated by acylCoA hydrolase causing a severe
organic acidaemia in rats (isovalerate plus 2-methylbutyrate up to
5 mM) I'II.
Inhibition of gluconeogenesis
Fatty acid oxidation may stimulate gluconeogenesis by providing
ATP, NADH where necessary, and
acetyl-CoA which is an obligatory
aUosteric effector for pyruvate
carboxylase (Ref. 14). This enzyme
is required for glucose synthesis
from pyruvate and lactate, and
alanine which must first be converted to pyruvate. Hypoglycin
(after a lag period) strongly inhibits the synthesis of glucose
from pyruvate, lactate and alanine,
but not significantly from glycerol,
dihydroxyacetone or fructose, in
isolated rat hepatocytes 14,zs, and
this inhibition might be explained
by inhibition of fatty acid oxidation. However, gluconeogenesis
is not dependent on fatty acid
oxidation when alternative substrates are available whose oxidation also provides ATP, NADH
and acetyl-CoA. These substrates
include pyruvate and lactate, and
the carbon skeletons of some
amino acids, which can be partitioned between oxidation and
glucose synthesis. MCPA-CoA
formed from hypoglycin in the
mitochondria causes a secondary
accumulation of isovaleryl-CoA, 2methylbutyryl-CoA, glutaryl-CoA
and butyryl-CoA. Elevated concentrations of these esters occur to
levels determined by their rates of
formation and of removal by
deacylation and by conjugation
with glycine and carnitine. These
acyl-CoA esters competitively inhibit the activation of pyruvate
carboxylase by acetyl-CoA and
this is probably the major mechanism of inhibition of gluconeogenesis 14.
Mechanism of whole-body
metabolic disturbances caused
by hypoglycin
It was suggested many years ago
that when fatty acid oxidation is

impaired by hypoglycin, increased

utilization of glucose and decreased gluconeogenesis lead to hypoglycaemia when hepatic glycogen
reserves are exhausted 3"4. Poisoning is also marked by ketosis with
a more oxidzzcd state of the
plasma acetoacetate-3-hydroxybutyrate ratio. Since hepatic ketogenesis is thought to be decreased
this may be due to greater inhibition of the peripheral utilization of
ketone bodies. The physiological
stress of hypoglycaemia mobilizes

free fatty acids from adipose tissue.

More long-chain acyl-CoA esters
are formed in the liver than can be
oxidized and some are used for
triacylglycerol synthesis with accumulation of neutral hepatic lipid.
Whole body energy production
may be impaired because of limited
fatty acid, ketone body oxidation,
and limited availability of glucose.
However, normal ATP concentrations are maintained in liver during
hypoglycin poisoning in rats (Ref.
16 and H. S. A. Sherratt, unpub-

mitochondria (10 mg of protein)

< palmitoyl-carnitine
(79)
430

~P~

;palmitoyl-carniline

oxygen uptake

T
400 n g - a t o m of 0 / ~
1
2min

Fig. 4. Experiment illustrating partial inhibition of mitochondrial ~-oxidatlon (from Ref. 9). A
rat liver mitochondrial fraction, 10 i~Mpaimitoyl-carnitine and 1.0 mM- MCPA were added
where indicated to 3.0 ml of medium at 30C containing 100 mM KCI, 2.5 mM phosphate,
5 mM MgCI2, 1.0 mM EDTA, 2.0 mM ADP (to maintain the maximum rate of electron
transport), tO mM malonata (to prevent oxidation of acetyl groups by the citrate cycle),
9 mg of defatted bowne serum albumin and 20 mM 3 (morpholino) propanesulphonate
(buffer), pH 7.2. Oxygen uptake was recorded potarographically. Rates of oxygen uptake
(ngatom O/mg of protein) are given in parenthesas, and the amount of oxygen consumed
by vertical arrows. PalmitoyI-CoA is germratdd in the mitochondrial matrix from added
palmitoyl-carnitine by the action of the camitine palmitoyltransferasas in the inner
membrane. In control incubations palmitoyI-CoA is quantitatively oxidized to acetoacetate
PalmitoyI-CoA + 702 - * 4Acetoacetate + CoASH
and endogenous respiration is largely suppressed during paimitoyl-camitine oxidation.
After incubation for 3 min with MCPA to allow formation of MCPA-CoA in the math'x, both
the rate and extent of oxidation of palmitoyI-CoA are decreased:
PalmitoyI-CoA + 602--~ 3Acetoacetate + Butyrate + CoASH
(see text). Similar results were obtained with even-chain acyl-carnitine asters (Ca to C14).
If MCPA was incubated with uncoupled mitochondria (in the presence of 0.2 pM
trifluormelhoxycarbonylcyanidephenylhydrazone, lOmM arsenate and 0.6mg of
valinomycin and absence of ADP) no inhibition was found of acyl-carnitine oxidation
indicating that prior A TP-dependent conversion of MCPA to MCPA-CoA is necessary.
Similar inhibitions are found in mitochondria prepared from rats which have been injected
with hypoglycin ~~.

190

TIPS - M a y 1986

Processes determining blood glucose concentrations

Net rate of [
1 Rateof
i n p u t o r [ absorption
output of = of dietary
glucose
glucose

[ 2 Rate of ]

3 Rateof
breakdown
of hepatic
andrenal
glycogen

Constant concentrations of blood glucose are maintained

when the rates of input and output into the circulation
are equal. Measurements of changes of glucose
concentration alone do not give the flux through the
glucose pool (see Refs 14, 17). (1) Zero during fasting; (2)
variable rate, occurs mainly in the liver but with some
contribution from the kidneys, stimulated by glucagon
and adrenaline; (3) free glucose is only derived from
glycogen in liver and kidney, stimulated by glucagon
and adrenaline; (4) mainly converted to CO2 and lactate;
lactate formed in extrahepatic tissues is used as precursor
for gluconeogenesis (Cori cycle). Glucose uptake by
some tissues (particularly muscle and adipose tissue) is
increased by insulin. Free fatty acids and ketone bodies
compete for oxidation and decrease glucose oxidation;
(5) occurs in both liver and many other tissues,
stimulated by insulin. Total extrahepatic glycogen
reserves are greater than in liver; (6) usually only occurs
in pathological conditions (diabetes) when blood
glucose concentrations exceed the renal threshold (about
10 mM). The rates of these processes, most of which are
under hormonal control, vary widely and change rapidly
after a meal or during and after exercise, and the liver can
switch rapidly between glucose uptake or output. The
system is normally self-adjusting and in the absence of
drugs or disease maintains glycaemia within desirable
limits. A normal fasting blood glucose concentration of
about 5 mM represents about 15 g of glucose in the
extracellular fluid of an adult human.
lished observations). ATP has not
been d e t e r m i n e d in other tissues so
it is not k n o w n if behavioural
depression and h y p o t h e r m i a are
caused b y i m p a i r e d s u p p l y of
metabolic energy or b y direct
pharmacologial effects of accumulated organic acids on the CNS, or
b y both. Behavioural depression
m a y decrease utilization of ATP so
that normal concentrations might
be m a i n t a i n e d even if the absolute
rate of oxidative phosphorylation
is impaired.
The above interpretation of the
effects of hypoglycin, although
plausible, is based on circumstantial evidence. Early attempts to
measure whole b o d y metabolism
b y administering ~4C-labelled fatty
acids or glucose to p o i s o n e d and
control animals and only measuring 14CO2exhaled gave no useful
information. The pool sizes of
substrates available to tissues are
changed b y hypoglycin, and further uncei'tainties are introduced

4 Rateof
utilization
of glucose

5 Rateof
glycogen ]
- synthesis in] liverand I
other
[
tissues ]

6 Rateof
[ excretion
ofglucose
I

The Scheme shows the requirement for acetyl-CoA,

NADH and ATP when pyruvate is converted to glucose
2Pyruvate + 4ATP + 2GTP + 2(NADH + H +)
---, Glucose + 4ADP + 2GDP + 6Pi + 2NAD +
Several steps and the control of the activities of
regulatory enzymes by hormones are not shown for
clarity.
[3-Oxidation in the mitochondrial matrix provides
acetyl-CoA required to activate pyruvate carboxylase,
and reducing equivalents for export to the cytosol to
reduce 1,3-diphosphoglycerate. B-Oxidation and oxidation of some of the acetyl-CoA formed by the citrate
cycle also provide NADH and ATP necessary for
gluconeogenesis. The oxidation of pyruvate and the
carbon skeletons of some amino acids may also provide
acetyl-CoA, NADH and ATP, although fatty acid
oxidation usually appears to be most important. Details
of the 'shuttles' transferring reducing equivalents, ATP
and oxaloacetate across the mitochrondrial inner
membrane are omitted. In hypoglycin-poisoning,
gluconeogenesis is impaired at the stage of the
conversion of pyruvate to oxaloacetate by pyruvate
carboxylase. The obligatory activation of pyruvate
carboxylase by acetyl-CoA is competitively inhibited by
unusual medium-chain acyl-CoA esters which accumulate in the matrix. A decreased availability of cytosolic
NADH may also contribute to the inhibition, although
ATP concentrations appear to be maintained.

b y h y p o t h e r m i a in small animals
(see Ref. 6). A n attempt was made
.to get quantitative information
about glucose metabolism b y a
kinetic study of the rate of decline
of the specific radioactivity of
blood glucose following a bolus
i.v. dose of [U-14C,2-3H]glucose
16 h after administration of hypoglycin (100 mg kg -1 body-wt) to
fasted rats (body temperatures
were artificially maintained) 17. Results indicated that gluconeogenesis was strongly inhibited
since recycling of glucose through
the Cori cycle (resynthesis of glucose in the liver from lactate formed
from glucose in peripheral tissues
b y glycolysis) was virtually abolished and the w h o l e - b o d y content
of free glucose was decreased b y
70%. The rate of utilization of
glucose was decreased b y 70%,
rather than increased as expected if
fatty acid oxidation is impaired. It
was concluded that hypoglycaemia
resulted from an even greater

i n h i b i t i o n of the rate of gluconeogenesis than of glucose utilization ~7.

It was also d e d u c e d that hepatic
glucose 6-phosphatase activity is
strongly i n h i b i t e d in vivo 14"17. It is
a mystery h o w poisoned rats
maintain even low concentrations
of blood glucose (2 mM) in the
apparent absence of glucose 6phosphatase activity14 since this
enzyme is thought to release
glucose into the blood from glucose 6-phosphate formed b y gluconeogenesis or b y glycogen breakdown.

Hypoglycin poisoning partly

resembles glutaric aciduria
Type II
Hypoglycin poisoning has many
similarities to some inborn errors
of metabolism involving defective
enzymes whose substrates are acylCoA esters 7'12. Rats given h y p o glycin provide the best model we
have for h u m a n hereditary glutaric

191

TIPS - M a y 1986

aciduria Type II where there is a

defect in the transfer of reducing
equivalents from all the mitochondrial acyl-CoA dehydrogenases, perhaps at the level of the
electron transferring flavoprotein
(ETP) or ETF dehydrogenase, to the
respiratory chain 7. This condition
is characterized by excretion of
isovaleric, 2-methylbutyric, isobutyric, butyric, some dicarboxylic
acids and isovaleryl-glycine. However, this resemblance is only
approximate since by contrast
with hypoglycin-poisoning where
palmitoyl-CoA dehydrogenase remains active, there is no hyperketonaemia.
A n t i d o t e s to hypoglycin p o i s o n i n g

It is remarkable that five different

compounds have each been reported to decrease the toxicity of
hypoglycin by different mechanisms.
G l u c o s e Early hopes that hypo-

glycin might be useful to treat

diabetes were soon a b a n d o n e d
because of its toxicity. Conversely,
administration of glucose can restore normal blood glucose concentrations but does not always
prevent death s .
R i b o f l a v i n Administration of ribo-

flavin partly protects against

chronic hypoglycin poisoning3.
Extra riboflavin may facilitate
replacement of chemically altered
flavin prosthetic groups of inhibited enzymes.
C a r n i t i n e Carnitine is an essential
cofactor for the m o v e m e n t of m a n y
acyl-groups across the i n n e r mitochondrial m e m b r a n e and is of
great current interest for the
treatment of some metabolic disseases TM. Bressler claimed in 1968
that administration of carnitine
decreased hypoglycin toxicity in
mice, which was an early therapeutic use of carnitine. However,
we tried unsuccessfully several
times to repeat this 6.
Glycine
Tanaka showed that
several branched-chain acyl-CoA
esters that are poor substrates for
the mitochondrial carnitine acyltransferases are conjugated with
glycine and MCPA-glycine, isovaleryl-glycine, a n d some butyrylglycine are excreted in hypoglycin
poisoning. The reaction between
glycine and acyl-CoA esters in the

mitochondrial matrix is catalysed

by glycine N-acylase which has a
low affinity for glycine (Kin =
3.3 mM) and tissue concentrations
of glycine are not saturating. Administration of large amounts of
glycine increases the rates of conjugation with glycine~9. This limits
the toxic effects of hypoglycin in
rats and minimizes the metabolic
disturbances, both decreasing enzyme i n h i b i t i o n by lowering tissue
concentrations of MCPA-CoA, and
by decreasing isovaleric acidaemia
by increased conversion of any
isovaleryl-CoA and still formed to
its inert glycine conjugate.
C l o f i b r a t e Clofibrate (ethyl pchlorophenoxyisobutyrate) is an
hypolipidaemic drug. When given
chronically in the diet (about 0.5%)
it causes a massive increase in the
n u m b e r of peroxisomes and of
peroxisomal [~-oxidation in the
livers of rodents such as rats and
mice, but not in most other species 2. Clofibrate also increases
mitochondrial B-oxidation in both
rodent and n o n - r o d e n t species.
Hypoglycin causes remarkable ultrastructural changes in the livers of
rats including large increases in
mitochondrial volume5. There was
also a 70% decrease in the n u m b e r
of peroxisomes 2. Hypoglycin was
therefore given to rats that had
been fed a diet containing 0.5%
clofibrate for 30 days. This caused
dramatic protection against the
toxic, hypoglycaemic and hypothermic effects and the animals
appeared normal. Coupled liver
mitochondria from clofibrate-fed
rats given hypoglycin oxidized
acyl-carnitine esters completely by
contrast with rats given hypoglycin
alone2. Curiously, uncoupled
mitochondria from both groups
only oxidized acyl-carnitine esters
as far as butyrate 2. Further,
butyryl-CoA dehydrogenase which
was inhibited by more than 90% in
hypoglycin-treated animals was
only inhibited by about 70% after
clofibrate feeding. This suggests
that clofibrate may induce an
additional butyryl-CoA dehydrogenase which is only active in
coupled mitochondria, perhaps as
a phosphorylated enzyme, and
which is not inactivated by MCPACoA.

[]

[]

[]

Hypoglycin poisoning iUustrates the complex metabolic dis-

turbances that can be caused by a

few primary enzyme inhibitions.
However, these disturbances are
still only partly understood after
three decades of investigation.
There have been several misunderstandings in interpretation6
and some unexpected turns in the
story 6"7"14'20.There is still no quantitative information on the perturbation of fatty acid, ketone body or
amino acid oxidation nor is it
k n o w n whether normal ATP concentrations can be m a i n t a i n e d in
extrahepatic tissues during hypoglycin poisoning. The abnormal
accumulation of acyl-CoA esters in
the mitochondrial matrix may
contribute more to the metabolic
disturbances than simple limitation of fatty acid oxidation. Many
problems remain and their elucidation will continue to give valuable information about both normal and abnormal metabolism.
References

1 Tanaka,K. (1972)J. Biol. Chem. 247,7465-7478

2 Mitchell,J. C. (1974)Diabetes 23, 919-920
3 Sherratt,H. S. A. (1969)Br. Med. Bull. 25,
250-255
4 Stewart, G. H. and Hanley,T. (1969)in
Oral Hypoglycaemic Agents (Campbell,
G. D., ed.), pp. 347-407,AcademicPress
5 Kean,E. A. (ed.) (1974)A Symposium on
Hypoglycin, AcademicPress
6 Sherratt, H. S. A. and Osmundsen, H.
(1976) Biochem. Pharmacol. 25, 743-750
7 Sherratt, H. S. A., Bartlett, K. and
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