The

International
Journal of
Biochemistry
& Cell Biology
PERGAMON The International Journal of Biochemistry & Cell Biology 30 (1998) 863±868

Molecules in focus

Carnosine, a protective, anti-ageing peptide?

Alan R. Hipkiss *
Molecular Biology and Biophysics Group, King's College London, Strand, London WC2R 2LS, UK
Received 25 September 1997; accepted 5 May 1998

Abstract

Carnosine (b-alanyl-L-histidine) has protective functions additional to anti-oxidant and free-radical

scavenging roles. It extends cultured human ®broblast life-span, kills transformed cells, protects cells against
aldehydes and an amyloid peptide fragment and inhibits, in vitro, protein glycation (formation of cross-links,
carbonyl groups and AGEs) and DNA/protein cross-linking. Carnosine is an aldehyde scavenger, a likely
lipofuscin (age pigment) precursor and possible modulator of diabetic complications, atherosclerosis and
Alzheimer's disease. # 1998 Elsevier Science Ltd. All rights reserved.

1. Introduction 3. Synthesis and degradation

Carnosine, ®rst identi®ed nearly a century Carnosine synthetase synthesises carnosine

ago, occurs in innervated tissues (e.g. muscle
and brain) of vertebrates at concentrations up
to 20 mM. Most published/patented items on
carnosine derive from Japan and the ex-Soviet
Union and indicate anti-oxidant roles. Recent
studies suggest additional homeostatic func-
tions.
from b-alanine and histidine in astroglial-rich pri-
mary cultures (rodent brain), glial cell lines and
skeletal muscle cell primary culture [2]. It is
degraded by serum or intracellular carnosinases,
but is resistant to peptidases that hydrolyse a-
peptides. The reason why carnosinase is in serum
is unknown, perhaps the relatively non-toxic
dipeptide is a histidine (relatively toxic) source in
the presence of carnosinase.

2. Structure 4. Biological function

Carnosine is a simple dipeptide, b-alanyl-L-
histidine (Fig. 1a).
* Tel.: +44-171-873-2490; fax: +44-171-873-2285.
Little is known about carnosine and metab-
olism; it may regulate glycolysis, muscular con-
traction and oxidative phosphorylation, stimulate
the immune system, bind copper, zinc and
calcium [23] and purines [19] which suggests

1357-2725/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved.

PII: S 1 3 5 7 - 2 7 2 5 ( 9 8 ) 0 0 0 6 0 - 0
864 A.R. Hipkiss / The International Journal of Biochemistry & Cell Biology 30 (1998) 863±868
potential involvement in gene regulation or signal
transduction.
4.1. Carnosine and ageing
Carnosine is one of the few agents that can
extend maximum cell division capacity (the
Hay¯ick limit) of cultured human ®broblasts
converting mature/senescent cells to juvenile
phenotypes [16]; it also decreases 8-hydroxy-
guanine (DNA oxidation product) content in cul-
tured human ®broblast DNA [13]. There have
been no reports (to date) on the e€ects of carno-
sine on ®broblast telomere length.
Ageing is associated with oxygen free-radical-
induced macromolecular damage; oxidised/cross-
linked/denatured proteins accumulate. Lysine
and histidine side chains are the most readily oxi-
disable groups in ageing proteins and carnosine
resembles both. Non-enzymic glycosylation (gly-
cation) of proteins is also an age-associated
phenomenon; sugar aldehydes react with protein
amino groups producing cross-linked, oxidised,
coloured, ¯uorescent, high molecular weight
structures called advanced glycosylation end-pro-
ducts (AGEs). Preferred glycation sites are amino
groups with proximal imidazole and/or carboxyl
groups; carnosine possesses these. Carnosine inhi-
bits, in vitro, formation of protein carbonyl
groups and cross-links, both age-associated modi-
®cations, by reacting preferentially with hexose,
pentose and triose aldehydes, malondialdehyde
(MDA, a lipid peroxidation product), methyl-
glyoxal (Hipkiss and Chana, unpublished obser-
vation) and ketones [6±8, 10] thus sparing
proteins. Whilst glycated lysine (lysine + glucose)
is mutagenic, glycated carnosine (carnosine +
glucose) is not [6]. Carnosine also prevents
acetaldehyde- and formaldehyde-induced DNA/
protein cross-linking [9].
Histidine forms spinacine following its reaction
with formaldehyde [20] (see Fig. 1b). Perhaps
carnosine forms analogous molecules with alde-
hydes (Fig. 1c) or even a tricyclic structure
(Fig. 1d). It is not known whether carnosine
reacts with protein carbonyls, but it is conceiva-
ble; in which case if carnosine's amino group
remains free (see Fig. 1c) it creates a quasi-lysine
residue and potential ubiquitination site for sub-
sequent proteolysis. Additionally/alternatively,
formation of AGE-like structures (possibly as
suggested in Fig. 1d) by carnosine reacting with
polypeptide carbonyl groups might facilitate
removal of modi®ed protein via receptors
(e.g. RAGEs) on scavenging macrophages.
Interestingly, growth with carnosine stimulates
degradation of long-lived proteins in cultured
human ®broblasts at high passage number [7].
Carnosine stimulates and inhibits calpain
activity (depending on calpastatin concen-
tration) [23], whilst in rabbit reticulocyte extracts,
it stimulates prematurely terminated (puro-
mycyl) peptide degradation, although inhibits
catabolism of normal length polypeptides con-
taining amino acid analogues perhaps because
of increased proteolysis of oxidised unlabelled
globin chains [9].
Carnosine is both a preventative and chain-
breaking anti-oxidant [3, 23] dismutating superox-
ide radicals, scavenging hydroxyl radicals and
preventing their production by binding copper
ions and interacting with lipid peroxidation pro-
ducts. Anti-oxidant activity is frequently
measured by MDA production (thiobarbituric
acid-reactive substances or TBARS); possibly,
direct anti-oxidant/radical scavenging function of
carnosine could be falsely ascribed because of its
reactivity towards MDA. Carnosine's anti-
oxidant role has been questioned by Aruoma et
al. [1] and Datta et al. [4].
Hypochlorite, generated from H2O2 and chlor-
ide ions by macrophage myeloperoxidase, induces
protein cross-linking and oxidation (formation of
carbonyl groups). Carnosine prevents hypochlor-
ite-mediated protein modi®cation in vitro [9] as it
reacts with hypochlorite to form a stable chlora-
mine derivative [3].
Carnosine protects cultured rat brain endo-
thelial cells against MDA toxicity [8], CHO cells,
human lymphocytes and ®broblasts against acet-
aldehyde and formaldehyde [7] and cultured
human ®broblasts and CHO cells against AGEs
formed by lysine/deoxyribose incubates [9].
Mammalian intramuscular carnosine concen-
trations increase with species maximal life-span
(Holliday, personal communication) though
 A.R. Hipkiss / The International Journal of Biochemistry & Cell Biology 30 (1998) 863±868 865

Fig. 1. (a) Structure of carnosine; (b) structure of spinacine, formed from the reaction of histidine with formaldehyde (formal-
dehyde carbon atom marked *); (c) hypothetical structure of product of carnosine's reaction with a (protein) carbonyl group (car-
bonyl carbon atom marked *) displaying quasi-lysine residue and (d) alternative hypothetical structure of product of carnosine's
reaction with a (protein) carbonyl group (carbonyl carbon atom marked *).

possibly decrease with age [12]. Long-lived non-
dividing tissues (muscle and nerve), rich in
carnosine, accumulate the age pigment lipofuscin,
conceivably deriving from carnosine's reaction
with MDA or other aldehydes. Although carno-
sine has also been suggested to possess immuno-
stimulatory activity, there are no published
reports on carnosine dietary supplementation on
animal life-span but carnosine feeding increase
vitamin E levels in rat tissues [4].
5. Possible medical and industrial applications
Some aberrant proteins associated with age-re-
lated pathologies (e.g. amyloid peptides and
AGE-proteins) induce hyperoxia, ROS and re-
lated products (e.g. MDA); protein carbonyls are
increased in neurodegenerative disorders [15].
Carnosine protects cultured rat brain endothelial
cells against MDA [8] and AGE-protein
and amyloid peptide fragment (b-A4 25±35)
866 A.R. Hipkiss / The International Journal of Biochemistry & Cell Biology 30 (1998) 863±868
Fig. 2. Summary of carnosine's known and possible activities and potential applications.
toxicity [9, 22]. Carnosine possibly: a€ects toxic
agent structure; interferes with AGE-receptors
(RAGEs) or with signal transduction; reacts with
ROS and end-products and stimulates aberrant
protein degradation.
Carnosine inhibits sugar-induced b-A4 amyloi-
dogenic peptide aggregation [18]. Alzheimer's
brain amyloid contains b-pleated sheets enriched
with iso- (or b)-aspartate [24], an age-associated
modi®cation which destabilise a-helices. Self-
selecting aggregation of `aged' peptides via b-
pleated sheets would be permissible between
chains containing iso-aspartates. Could carno-
sines (b-peptides) hydrogen bond to iso-aspartate
residues and disrupt `aged' polypeptide aggrega-
tion?
Little is known about age-related changes in
carnosine concentrations in either normal or
Alzheimer's brain. The carnosine content of the
olfactory lobe is high; loss of a sense of smell is
an assumed early indicator of A.D. Age-related
declines in CSF homocarnosine [21] and carno-
sine and anserine in rat skeletal and cardiac
muscle are reported [12]. Carnosine prevents glu-
tathione oxidation in vitro, its concentrations in
human lens declines, together with glutathione,
with increasing cataract severity [3]. Carnosine
inhibits cataractogenesis and atherosclerotic fatty
streaks in rabbits fed high cholesterol diets [17].
Atherosclerosis is associated with protein modi®-
cations possibly induced by hypochlorite and
MDA, against which carnosine is protective (see
Section 4.1).
5.1. Carnosine as an anti-cancer agent
Carnosine selectively kills cultured transformed
cells; however this is prevented by pyruvate,
oxaloacetate and a-ketoglutarate [11]. Another
b-peptide, b-alethine (b-alanyl-cysteamine disul-
phide) has anti-neoplastic activity and extends
cultured human ®broblast life-span [14]. Patent
 A.R. Hipkiss / The International Journal of Biochemistry & Cell Biology 30 (1998) 863±868
claims suggest that carnosine and bestatin, an
carnosinase inhibitor, are useful therapeutically
as anti-tumour agents.
5.2. Potential therapeutic applications
Carnosine, or related molecules less susceptible
to serum carnosinase, e.g. carcinine, could be
explored [17] for its therapeutic potential to con-
trol secondary complications in diabetes (catarac-
togenesis and atherosclerosis) because of the
dipeptide's ability to intervene in protein glyca-
tion, AGE production and reactivity. Because it
modulated amyloid peptide toxicity [22] carnosi-
ne's therapeutic potential in Alzheimer's disease
should be investigated (and perhaps in¯amma-
tory conditions in general) where both oxidation
and glycation phenomena are possibly involved.
Additionally carnosine's use in control of tumour
growth in combination with bestatin should be
studied.
Finally, should we eat carnosine-containing
food (i.e. meat) when drinking alcoholic bev-
erages, given carnosine's ability to inhibit acet-
aldehyde-mediated damage? Alternatively, should
brewers, etc. include carnosine in their products
to protect our livers? The properties (actual and
potential) and possible applications of carnosine
are illustrated in Fig. 2.
Acknowledgements
I thank Robin Holliday for bringing carnosine
to my attention and for his hospitality in
Australia and Geo€rey Grigg for persuading
Robin to take an interest in this remarkably non-
toxic molecule.
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