BASIC NEUROSCIENCES, GENETICS AND IMMUNOLOGY - ORIGINAL ARTICLE

From caffeine to fish waste: amine compounds present in foodand drugs and their interactions with primary amine oxidase

Aldo Olivieri • Daniel Rico • Zhied Khiari •

Gary Henehan • Jeff O’Sullivan • Keith Tipton

Received: 29 September 2010 / Accepted: 16 February 2011

� Springer-Verlag 2011

Abstract Tissue bound primary amine oxidase (PrAO)

and its circulating plasma-soluble form are involved,

through their catalytic activity, in important cellular roles,

including the adhesion of lymphocytes to endothelial cells

during various inflammatory conditions, the regulation of

cell growth and maturation, extracellular matrix deposition

and maturation and glucose transport. PrAO catalyses the

oxidative deamination of several xenobiotics and has been

linked to vascular toxicity, due to the generation of cyto-

toxic aldehydes. In this study, a series of amines and

aldehydes contained in food and drugs were tested via a

high-throughput assay as potential substrates or inhibitors

of bovine plasma PrAO. Although none of the compounds

analyzed were found to be substrates for the enzyme, a

series of molecules, including caffeine, the antidiabetics

phenformin and tolbutamide and the antimicrobial pent-

amidine, were identified as PrAO inhibitors. Although the

inhibition observed was in the millimolar and micromolar

range, these data show that further work will be necessary

to elucidate whether the interaction of ingested biogenic or

xenobiotic amines with PrAO might adversely affect its

biological roles.

Keywords Primary amine oxidase � Semicarbazide-

sensitive amine oxidase � Xenobiotic(s) � Enzyme

inhibition

Abbreviations

PrAO Primary amine oxidase

SSAO Semicarbazide-sensitive amine oxidase

TPQ 3,4,5-Trihydroxyphenylalanine quinone

DAO Diamine oxidase

Introduction

Primary amine oxidase (PrAO) is the accepted name for the

copper-containing enzyme (EC 1.4.3.21) that catalyses the

oxidative deamination of endogenous and exogenous pri-

mary amines, according to the overall reaction:

RCH2NH2 þ O2 þ H2O �!PrAORCHOþ NH3 þ H2O2

It was previously classified with a group of other enzymes

as semicarbazide-sensitive amine oxidase (SSAO), EC

1.4.3.6 (Boyce et al. 2009). The sensitivity to inhibition by

carbonyl-group reagents, such as semicarbazide, is a result of

the presence of a 6-hydroxydopa: 2,4,5-trihydroxyphen-

ylalanine quinone (TPQ) residue as the redox cofactor. The

enzyme is active towards aliphatic and aromatic primary

amines but secondary amines, such as adrenaline are not

oxidized. The important physiological substrates are

believed to include methylamine and aminoacetone (see

O’Sullivan et al. 2004).

A. Olivieri (&) � K. Tipton

School of Biochemistry and Immunology, Trinity College,

Dublin 2, Ireland

e-mail: oliviera@tcd.ie

D. Rico � Z. Khiari � G. Henehan

Dublin Institute of Technology, School of Food Science and

Environmental Health, Cathal Brugha Street, Dublin 1, Ireland

J. O’Sullivan

Dublin Dental School and Hospital, Trinity College,

Dublin 2, Ireland

Present Address:A. Olivieri

Department of Pharmacology, University of Alberta,

9-70 Medical Sciences Building, Edmonton,

AB T6G 2H7, Canada

123

J Neural Transm

DOI 10.1007/s00702-011-0611-z

PrAO is a membrane-bound protein in mammalian

species (Lewinsohn 1984), although a plasma soluble form

of the enzyme also exists, which results from the proteo-

lytic cleavage of membrane-bound PrAO (Stolen et al.

2004; Ekblom et al. 2000). The amount of the plasma form

varies greatly between species. Although it is normally low

in the human, it has been shown to be increased in a

number of disease states (see O’Sullivan et al. 2004). The

products of the PrAO catalysed reaction, especially

hydrogen peroxide, have important cellular roles, including

the regulation of cell growth and maturation, extracellular

matrix deposition and maturation and glucose transport

capacity. It has been demonstrated that the generation of

H2O2 during the oxidation of amine substrates by PrAO

stimulates glucose uptake by the recruitment of insulin

receptors to the cell-surface (for review, see O’Sullivan

et al. 2004). In some tissues, during pathological insult,

PrAO functions as a vascular adhesion protein, VAP-1, that

mediates the slow rolling and adhesion of lymphocytes to

endothelial cells, contributing to their transmigration into

sites of inflammation (Jalkanen and Salmi 2001). H2O2

generated by substrate oxidation also appears to be nec-

essary for this adhesion process to occur (O’Sullivan et al.

2007; Jalkanen et al. 2007; Olivieri and Tipton 2011).

Since all these processes involve the catalytic activity of

the enzyme, they are inhibited by PrAO inhibitors. They

have also been shown to be attenuated by knock-out of the

PrAO (AOC3) gene (Bour et al. 2007; Stolen et al. 2005).

The active site of the tissue-bound PrAO is located in

the extracellular domain (Jakobsson et al. 2005) making the

enzyme, together with its circulating plasma-soluble form,

a scavenger of potentially toxic amines in the blood. Food

products may contain a wide variety of biogenic amines

(see Table 1), which are often a consequence of enzymatic

decarboxylation (by microorganisms) of the corresponding

amino acids. Such amines may be sources of nitrogen and

Table 1 A selection of amines and amine derivatives that may be ingested and their pharmacological/toxic effects

Amine Sources Adverse effects

Histamine Fish, fermented sausages, cheese,

alcoholic drinks

Asthmatic attack, wheezing, decreased blood pressure,

urticaria, gastrointestinal symptoms, headache

Tyramine Cheese, pickled herring, chicken liver,

alcoholic drinks

Peripheral vasoconstriction, increases the cardiac output,

causes lacrimation and salivation, increases respiration,

increases blood sugar level, releases noradrenaline from

the sympathetic nervous system

2-phenylethylamine Chocolate, cheese, meat products Releases noradrenaline from the sympathetic nervous

system, increases the blood pressure, headache

Serotonin (5-hydroxytryptamine) Fruit (e.g. bananas) Inhibition of gastric secretion, stimulation of smooth

muscle, heart lesions (endomyocardial fibrosis, possibly)

Dopamine Bananas and other fruit, some vegetables Cardiotonic, antihypotensive (hypertensive crisis),

antioxidant and pro-oxidant

Tryptamine Cheese, meat and meat products,

fermented vegetables

Increases blood pressure

Methylamine Tobacco smoke (vapour phase), major

end-product of nicotine metabolism

Relatively non-toxic in the absence of SSAO

Allylamine Environment (used in the manufacture of

pharmaceuticals and vulcanized rubber)

Cardiovascular lesions

Volatile aliphatic amines Tobacco smoke (vapour phase)

Aromatic amines Tobacco smoke (particulate phase)

Nitrosamines Tobacco smoke (vapour and particulate

phase), salted, pickled and fermented

foods

Carcinogenic

Heterocyclic amines Cooked foods (cooking method) Carcinogenic

Spermine and spermidine Ubiquitous in food (e.g. fish, meat and

vegetable products)

Cell proliferation and differentiation

Cadaverine Fish, cheese, meat and meat products,

fermented vegetables

Hypotension, bradycardia, lockjaw, paresis of the

extremities, potentiates the toxicity of other amines

Putrescine Ubiquitous in food (e.g. fish) Cell proliferation and differentiation, hypotension,

bradycardia, lockjaw, paresis of the extremities,

potentiates the toxicity of other amines

Agmatine Fish, meat and vegetable products Modulation of pain, anticonvulsive effects

Adapted from Strolin Benedetti et al. (2007)

A. Olivieri et al.

123

are precursors for the synthesis of hormones, alkaloids,

nucleic acids and proteins, however, they can also pose

toxicological risks (KaroviCova and Kohajdova 2003;

Boobis et al. 2009). Because of microbiological decarbox-

ylation, biogenic amines are present in fermented products

(e.g. cheese 5–4500 mg kg-1, wine 5–130 mg dm-3, beer

2.8–13 mg dm-3, sauerkraut 110–300 mg kg-1) and in

improperly kept food (such as fish 2400–5000 mg kg-1,

beef liver about 340 mg kg-1, prepared meats 10–700

mg kg-1), (KaroviCova and Kohajdova 2003).

PrAO also catalyses the oxidative deamination of a

number of xenobiotics, including mescaline and the anti-

malarial drug primaquine (see O’Sullivan et al. 2004;

Tipton and Strolin Benedetti 2001). Metabolism of the

xenobiotic allylamine by PrAO produces acrolein, which

causes vascular toxicity (Hysmith and Boor 1988).

A common source of xenobiotic amines would be

through intake of drugs and additives present in food.

A number of antidiabetic drugs contain amino groups.

Metformin (dimethylbiguanide, see Fig. 2) is among the

most widely prescribed agents in the treatment of diabetes.

A related compound, aminoguanidine, still used as an

antidiabetic, has been reported to be a potent PrAO

inhibitor (Yu and Zuo 1997; Prevot et al. 2007). The anti-

inflammatory methylguanidine (Fig. 2) is an inhibitor of

diamine oxidase, DAO, another member of the SSAO

family of enzymes (Gang et al. 1976). Other guanidine

compounds include 1-phenethylbiguanide hydrochloride

(phenformin), which was used as antidiabetic, although its

use was discontinued for toxicity problems, 1-butylbigua-

nide (butformin), still used as antidiabetic, and the anti-

microbial pentamidine (see Fig. 2 for chemical structures

of phenformin, butformin and pentamidine). Phenformin

and pentamidine were also reported to inhibit DAO (Cubrıa

et al. 1991).

These and other dietary nitrogenous compounds, such

as caffeine, in tea, coffee, soft drinks and the now

popular ‘energy’ drinks, were assayed in this work for

their possible interaction with PrAO (see also Olivieri

and Tipton 2011). Some common dietary aldehydes

including vanillin, syringaldehyde, salicylaldehyde, cyn-

namaldehyde and anisaldehyde were also tested for any

potential interaction with the enzyme (see Figs. 1a, b and

2 for structures).

Given the multi-functionality of PrAO, especially in

relation to its roles in cell adhesion, maturation and glucose

uptake, it is important to know whether possible interactions

with biogenic and xenobiotic amines might occur after die-

tary intake or administration as drugs, since they might affect

the physiological roles of the enzyme. This work was

designed to test the inhibition of PrAO by a variety of such

compounds, as listed in Tables 2, 3 and 4, in order to gain

insights into possible interactions and to help identify lead

compounds for future drug developments.

Materials and methods

Sources of reagents

Bovine plasma PrAO was obtained by the supplier BioVar

Ltd, Yerevan, Armenia. Other enzymes and chemicals used

in this study were obtained from Sigma-Aldrich, unless

otherwise indicated.

Enzyme assay procedures

The activity of PrAO was determined following the pro-

duction of H2O2 at 498 nm, by the method of Holt and

Palcic (2006), in the presence of 5 mM benzylamine and

10 lg/ml PrAO. The chromogenic solution for the detec-

tion of H2O2 contained 1 mM vanillic acid, 0.5 mM

4-aminoantipyrine and horseradish peroxidase (4 U/ml) in

a ‘physiological’ HEPES buffer system (100 mM HEPES,

280 mM NaCl, 10 mM KCl, 4 mM CaCl2, 2.8 mM

MgCl2). The pH of the buffer was adjusted to 7.4 with

0.1 M NaOH. Assays were completed in a reaction volume

of 300 ll in 96-well microtitre plates, at 37�C, using a

SpectraMax 340PC plate reader (Molecular Devices, Inc.

Sunnyvale, CA 94089-1136, USA).

Control assays of the coupling system, in the pres-

ence of 10 lM H2O2, 1 mU/ml HRP but in the absence

of PrAO, showed that none of the compounds affected

the peroxidase chromogenic detection system. Each

compound was assayed in triplicate, at a final con-

centration of 1 mM and after a preincubation time of

0 min or 60 min, at 37�C, before adding substrate. If

any compound was found to show substantial PrAO

inhibition at 0 min and/or after 60 min of preincuba-

tion, it was further analyzed at different concentrations

and/or different times of preincubation to elucidate the

inhibition type.

Inhibition of bovine plasma PrAO in the presence

of protein hydrolysates from fish-waste sources

The samples shown in Table 5 were obtained from mack-

erel viscera, bones and gelatin after hydrolysis with dif-

ferent peptidases. All samples were diluted to a working

concentration of 1 mg/ml in assay buffer and added at 0, 60

and 120 min to the reaction mixture, for pre-incubation at

37�C, before adding substrate. Although the proteolysis

was completed at various pH values (see Table 5), control

experiments showed that when the compounds were diluted

From caffeine to fish waste

123

at 1 mg/ml in buffer the pH was 7.4 at their assay

concentrations.

Kinetics of Inhibition

IC50 values were determined by plotting the initial rates,

expressed in A498 nm 9 10-3 min-1 vs log [inhibitor] and

fitting the resulting plot to a sigmoidal curve, whose

point of inflection represents the log IC50 value for the

inhibitor studied. The initial rates (v = abs498 nm 9

10-3 min-1) of H2O2 formation were determined at 37�C

and pH 7.4. Data were then fitted to the Michaelis–

Menten equation by non-linear regression and the value

of Ki was determined from the dependence of the kinetic

parameters on the inhibitor concentration. The curves

were fitted with the aid of the computer software

GraphPad Prism, version 5.00.

Statistical analysis

Data collected were the mean values ± S.E.M. of at least

three independent experiments. Two-way ANOVA and

Bonferroni post tests were completed with the aid of the

computer software GraphPad Prism, version 5.00. Differ-

ences between two groups of data were considered

statistically significant when comparison tests resulted in

two-tailed P value \ 0.05.

Results

Effects of caffeine and polysaccharides

With the exception of caffeine, none of the compounds

shown in Table 3 showed significant inhibition of PrAO.

Fig. 1 The structures of a

selection of amines and

aldehydes present in food

(a) and nutraceuticals (b) that

were screened for interactions

with PrAO

A. Olivieri et al.

123

All were screened at concentrations of 1 and 10 mM,

except chondroitin sulphates A and B, which were tested in

the concentration range of 1–5 mg/ml and chitosan, where

a range of 0.1–10 mg/ml was used. It was not possible to

assess the effects of chitin, because of its insolubility.

Caffeine was an effective inhibitor with an IC50 of

0.8 ± 0.3 mM, as shown in Fig. 3. The inhibition, which

was not time dependent, was previously shown of a mixed-

type inhibitor with respect to methylamine, with a Ki and

Ki0 values of approximately 1.0 and 8.0 mM (Olivieri and

Tipton 2011).

Screening of common antidiabetic drugs as potential

inhibitors of PrAO

The compounds screened as potential PrAO inhibitors and

the concentration ranges used in the assays are listed in

Table 6. Phenformin (Fig. 4) was shown to inhibit PrAO

weakly (IC50 = 26.7 ± 1.1 mM), with no time depen-

dence observed when the enzyme was preincubated with

inhibitor for 1 h prior to the addition of substrate. Tolbu-

tamide was also a weak PrAO inhibitor, causing 20%

inhibition at a concentration of 1.0 mM, with no time

dependence. Butformin, metformin and methylguanidine

were not found to inhibit PrAO in the range 0–10 mM.

Phenytoin, aminotriazole and pentamidine and their

potential interactions with PrAO

The anti-epilectic drug phenytoin did not inhibit PrAO at

concentrations up 1 mM and preincubation for up to 1 h.

The weed killer aminotriazole was a weak inhibitor (ca.

20%) of the enzyme when used in the concentration range

0–1.0 mM and incubation times up to 1 h. No time

dependence was observed (see Table 7). However, the

antimicrobial drug pentamidine was found to inhibit PrAO

in the range 0.1–100 lM, with IC50 *3.5 lM with no

time-dependence observed (Fig. 5).

Inhibition of bovine plasma PrAO by protein

hydrolysates from fish-waste sources

14 samples containing protein hydrolysates from mackerel

bones, viscera and gelatin (see Table 5 for more informa-

tion on the samples) were assayed as potential PrAO

inhibitors, with significant findings that may be summa-

rized as follows (see Fig. 6a, b):

Samples 1 and 2 (from mackerel head and skin gelatin,

respectively, hydrolysed with pepsin) showed approxi-

mately 15% inhibition of PrAO at 0.25 mg/ml when

assayed without preincubation with the enzyme. This

inhibition increased to about 50% after incubation for 1 or

2 h prior to the addition of benzylamine.

Samples 7 and 8 (from mackerel viscera, hydrolysed by

endogenous proteases and with Aspergillus oryzae prote-

ase, respectively) behaved similarly to samples 1 and 2 at

time zero when present in the assayed without preincuba-

tion with the enzyme. Inhibition of PrAO was time

dependent, with approximately 30 and 50% inhibition after

incubation with the enzyme for 1 and 2 h, respectively,

before adding benzylamine.

Samples 11 and 12 (from mackerel viscera, hydrolysed

by trypsin and chymotrypsin, respectively) showed inhi-

bition of PrAO after 1 and 2 h of preincubation with the

enzyme prior to the addition of substrate. However, they

did not show any inhibition of PrAO at time zero.

None of the other hydrolysates tested had any significant

effects on PrAO activity.

Discussion

Determination of hydrogen peroxide formation indicated

that none of the compounds studied were substrates for

PrAO in the concentration ranges used. The ‘‘food addi-

tives’’ analysed were found to have no significant effects

on PrAO activity under the conditions used in this work.

Although substrate oxidation by PrAO yields an aldehyde

product and, therefore, product inhibition might be

Fig. 1 continued

From caffeine to fish waste

123

expected, the inhibition of the enzyme from pig plasma by

benzylaldehyde was reported to be competitive with

respect to benzylamine with a Ki of about 0.7 mM for the

enzyme from pig plasma (Taylor et al. 1972), none of the

aldehydes investigated had a significant effect on PrAO

activity. Neither the sweetener aspartame nor the flavour

enhancer monosodium glutamate inhibited PrAO. The

failure of 6-aminocaproic acid to inhibit was interesting,

since this can be regarded as an a-deaminated analogue of

L-lysine, which was reported to be an inhibitor of the

enzyme (Olivieri et al. 2007, 2010). The failure of chitosan,

which is widely used as an aid to slimming (see Jull et al.

2008) and the chondroitin sulphates to inhibit PrAO con-

trasts with the behaviour of free aminosugars, which have

been shown to inhibit the enzyme O’Sullivan et al. 2007).

Caffeine, which is probably among the most commonly

ingested drugs, since it is present in coffee, tea, numerous

soft drinks and ‘energy’ drinks has been shown to be a

PrAO inhibitor. Caffeine is a purine, containing an imid-

azole ring, thus it is possible that it might bind to an

inhibitory imidazoline binding site (I2) on PrAO (see Mu

et al. 1994; Holt et al. 2008; Olivieri and Tipton 2011). A

Fig. 2 The structures of a

selection of antidiabetic, anti-

epileptic and antimicrobial

drugs screened for interactions

with PrAO in this study

A. Olivieri et al.

123

‘‘typical’’ cup of coffee (5 oz) contains 40–170 mg of

caffeine (depending on how the coffee is brewed). Around

40 mg are contained in the average cup of tea (5 oz), a can

of Coke (12 oz) and in an espresso shot (1 oz). Some

‘energy’ drinks contain a higher quantity of caffeine,

varying from 80 to 300 mg. Caffeine is easily absorbed

into the bloodstream from the digestive tract and easily

moves out of the blood and into the tissues. The half-life

for caffeine in the bloodstream is about 3–4 h, while its

maximum concentration is achieved in 0.5–1 h after

ingestion (Klosterman 2006). Momoi et al. (2008) reported

that the ingestion of a regular cup of coffee corresponds to

a caffeine blood concentration of 5–10 lM, 30 min after

consumption. Battram et al. (2006) showed that acute

administration of 4.5 mg/kg (a regular cup of coffee would

provide about 1–2 mg/kg of caffeine) to 10 non-caffeine

users and 1 caffeine user of average body weight of 76 kg

resulted in a blood caffeine concentration that peaked at

36.8 ± 1.5 lM. These values remain well below the IC50

of 0.8–1.0 mM calculated in this work for the inhibition of

PrAO by caffeine. However, it cannot be excluded that

consumption of high concentrations of caffeine in a short

time might lead to significant physiological levels of PrAO

inhibition. Since PrAO is involved in regulating the uptake

of glucose (Enrique-Tarancon et al. 2000; El Hadri et al.

2002; Morin et al. 2002; Zorzano et al. 2003) it would be

interesting to investigate whether small, chronic levels of

PrAO inhibition among subjects who use caffeine in excess

might affect glucose uptake. Further work will be neces-

sary to show whether this inhibition of PrAO has any

physiological implications.

Several other amine compounds, commonly used as

antidiabetic drugs, were investigated in this work. 1,1-

Dimethylbiguanide HCl (metformin), one of the most

commonly used drugs in the treatment of diabetes and

2-butyl-1-(diaminomethylidene)-guanidine (butformin) did

not result in any significant PrAO inhibition. However,

phenformin (1-phenethylbiguanide�HCl) and tolbutamide

both acted as weak PrAO inhibitors. Such inhibition is

unlikely to be significant at the plasma levels reported after

administration of these antidiabetic drugs (see Marchetti

et al. 1987). Other compounds screened included the

antiepileptic drug diphenyldantoin (Phenytoin), which did

not inhibit PrAO and the weed-killer aminotriazole (con-

sisting of a pyrrolic ring and a primary amino group),

which acted as PrAO inhibitor, albeit a poor one.

The most potent inhibitor found among all the com-

pounds screened for this work was the antimicrobial

drug pentamidine. Pentamidine (1,5-bis(4-amidinophenoxy)

pentane) is a diamidine that has been reported to be a DAO

inhibitor and is used to treat pneumocystis carinii pneu-

monia (an infection common among immunosuppressed

patients; see Hughes et al. 1978). The fact that it is also an

inhibitor of PrAO from this source was surprising since not

many diamines display this property. Other polyamines,

such as spermidine, were reported to be able to bind to

PrAO at both the active site and a second binding site close

to the TPQ cofactor (Holt et al. 2008). It cannot be

excluded that pentamidine might bind to PrAO in a similar

fashion. Furthermore, the aromatic rings in proximity to the

amino groups of the molecule (see Fig. 2) might play a role

in the binding. The IC50 value for pentamidine was similar

to the maximum concentration of this drug reported in

human plasma after a standard 4 mg/kg dose administered

by intramuscular injection (Sands et al. 1985). Therefore,

Table 3 Some amines and aldehydes, contained in food and addi-

tives, that were screened as potential inhibitors of PrAO in this study

Aldehydes: vanillin,

syringaldehyde, cynnamaldehyde

Food flavourings

Aspartame Sweetener

Caffeine Coffee, tea, soft drinks

Monosodium glutamate Meat, food additive

Chitin, chitosan Vegetable, etc., products

6-Aminocaproic acid and

7-aminoheptanoic acid

From lysine metabolism

Trimethylamine oxide Fish

Chondroitin sulphate A and B Nutraceuticals, used for the

treatment of ostheoarthritis

Polyamines: putrescine,

spermidine, spermine and

cadaverine

Seafoods, etc.

Table 4 Amine compounds that were considered potential PrAO

inhibitors

Phenytoin (diphenyldantoin) Anti-epileptic

Aminotriazole Weed killer, carcinogen

Pentamidine Antimicrobial

Table 2 A selection of common antidiabetic drugs that were

screened as potential inhibitors of PrAO in this study

1,1-dimethylbiguanide HCl (metformin)—in common use

1-Phenethylbiguanide HCl (phenformin, phenethylbiguanide HCl)—

discontinued for toxicity problemsa

1-Butylbiguanide (butformin, 2-butyl-1-(diaminomethylidene)-

guanidine)—still commonly used

Aminoguanidine—reported to be antidiabetic and anti-ageingb

Methylguanidine—anti-inflammatory also formed in vivoc

Tolbutamide—one of the sulfonylurea group of drugs

a Phenformin is reported to inhibit diamine oxidase (Cubrıa et al.

1991)b Known to inhibit SSAO (Yu and Zuo 1997)c Inhibits DAO (Gang et al. 1976)

From caffeine to fish waste

123

a transient inhibition of PrAO might be expected after

administration of this drug.

The series of hydrolysates from mackerel gelatin (from

head and skin), viscera and frames were prepared using

different proteolytic enzymes under various conditions of

time and pH. Folador et al. (2006) showed that hydroly-

sates from the major fish wastes (heads, viscera, skin and

skeleton) contained approximately 5% lysine (of the total

amino acidic content), along with variable quantities of

biogenic amines, including histamine, tryptamine, putres-

cine, cadaverine, spermine, spermidine and tyramine.

Although these may make some contribution to the inhi-

bition observed, it was found that the nature of the pepti-

dase was critical for the formation of hydrolysates that

contained PrAO inhibitors. When gelatin from head and

skin was digested in pepsin, the hydrolysates produced

were found to inhibit PrAO in a time-dependent manner,

but this was not the case when the same hydrolysis was

carried out with trypsin or chymotrypsin. When the source

was mackerel viscera, the opposite occurred with trypsin

and chymotrypsin releasing material capable of inhibiting

PrAO after 1 and 2 h (s) of preincubation with the enzyme.

No PrAO inhibition was observed when the same hydrol-

ysates were digested in pepsin. The most effective prote-

olytic activities for mackerel viscera, in terms of generation

of PrAO inhibitors, were as a result of endogenous protease

activity (autolysis) and the protease preparation from

Table 5 Sources of protein

hydrolysates from fish-wasteSample

number

Source Enzyme used for

digestion

Hydrolysis

time (h)

pH Protein content

(mg/ml)

1 Head gelatine Pepsin 24 2.03 5.11

2 Skin gelatine Pepsin 24 2.02 4.96

3 Head gelatine Trypsin 24 7.59 5.04

4 Skin gelatine Trypsin 24 7.31 12.84

5 Head gelatine Chymotrypsin 24 7.7 3.68

6 Skin gelatine Chymotrypsin 24 7.59 11.48

7 Viscera Endogenous 24 6.97 2.36

8 Viscera Protease from

Aspergillus oryzae24 7.15 2.21

9 Viscera Protease from

Bacillus licheniformis24 7.2 6.82

10 Viscera Pepsin 24 2.95 7.17

11 Viscera Trypsin 24 7.2 3.01

12 Viscera Chymotrypsin 24 7.21 4.85

13 Frames Protease from

Aspergillus oryzae4 7.23 7.28

14 Frames Protease from

Bacillus licheniformis4 6.87 11.91

Fig. 3 The inhibition of PrAO by caffeine. The initial rates

(v = abs498 nm 9 10-3 min-1) of formation of H2O2 during the

oxidation of benzylamine by PrAO, were determined, at 37�C and pH

7.4, in the presence of the indicated concentrations of caffeine. The

curve fit and the value of IC50 = 0.8 ± 0.3 mM were obtained with

the aid of the computer software GraphPad Prism, version 5.00. The

x-axis is shown in logarithmic scale. All data were obtained from at

least three independent experiments, each completed in triplicate

Table 6 Screening of potential PrAO inhibitors among some com-

mon antidiabetic drugs

Compounds Concentration

range (mM)

Inhibition

1,1-dimethylbiguanide

HCl (Metformin)

0–10 None detected

1-Butylbiguanide

(butformin)

0–10 None detected

Methylguanidine 0–10 None detected

1-Phenethylbiguanide

HCl (Phenformin)

0–100 Yes

IC50 = 26.7 ± 1.1 mM

Tolbutamide 0–1 Yes

20 ± 1% at 1 mM

A. Olivieri et al.

123

Aspergillus oryzae. In contrast, the protease activity from a

Bacillus licheniformis preparation was ineffective. These

findings indicate that the effective inhibitors are peptides

generated by enzymes of appropriate specificities.

Although the specificities of chymotrypsin, pepsin and

trypsin are well known, the same cannot be said of the

peptidases from Aspergillus oryzae and Bacillus licheni-

formis, which appear to be complex mixtures of more than

one enzyme. Autolytic digestion is more complex in terms

Fig. 4 The inhibition of PrAO by 1-phenethylbiguanide�HCl (phen-

formin). The initial rates (v = abs498 nm 9 10-3 min-1) of formation

of H2O2, developed during the oxidation of benzylamine by PrAO,

were determined, at 37o C and pH 7.4, in the presence of the indicated

concentrations of phenformin. The curve fit and the value of

IC50 = 26.7 ± 1.1 mM were obtained with the aid of the computer

software GraphPad Prism, version 5.00. The x-axis is shown in

logarithmic scale. All data were obtained from at least three

independent experiments, each completed in triplicate. Error barsnot evident were smaller than the experimental points

Table 7 Screening of potential PrAO inhibitors among some drugs

and pesticides

Compounds Concentration

range

Inhibition

Phenytoin (diphenyldantoin) 0–1 mM None detected

Aminotriazole 0–1 mM Yes

20 ± 1% at 1 mM

Pentamidine 0–100 lM Yes

IC50 = 3.5 ± 0.50 lM

Fig. 5 The inhibition of PrAO by the antimicrobial pentamidine. The

initial rates (v = abs498 nm 9 10-3 min-1) of formation of H2O2,

formed during the oxidation of benzylamine by PrAO, were

determined, at 37�C and pH 7.4, in the presence of the indicated

concentrations of pentamidine. The curve fit and the value of

IC50 = 3.5 ± 0.50 lM were obtained with the aid of the computer

software GraphPad Prism, version 5.00. The x-axis is shown in

logarithmic scale. All data were obtained from at least three

independent experiments, each completed in triplicate. Error barswere smaller than the representation of the point

Fig. 6 Inhibition of bovine plasma PrAO in the presence of protein

hydrolysates from various sources. For information regarding the

nature of the samples refer to Table 7. All the samples were diluted to

a working concentration of 1 mg/ml in assay buffer. The final

concentration of the hydrolysates in the assay was 0.25 mg/ml. The

initial rates (v = abs498 nm 9 10-3 min-1) of formation of H2O2,

formed during the oxidation of benzylamine by PrAO, were

determined, at 37�C and pH 7.4. The data shown in the figure are

the mean values ± S.E.M. of three independent experiments, each

completed in triplicate. Statistical analysis (two-way ANOVA) was

completed with the aid of the computer software Prism, version 5.0.

Effect due to different samples P \ 0.0001, effect due to the time of

preincubation P \ 0.0001, interaction effect P \ 0.0001. The differ-ent columns were compared to their respective control (Ctrl) via

Bonferroni post test. *P \ 0.05, **P \ 0.001, ***P \ 0.0001

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of the enzymatic processes involved. The possibility that

amine formation, resulting from decarboxylation of any

free amino acids released during the process, might con-

tribute to the inhibition observed in these samples cannot

be excluded in this case. High levels of some amines can

also arise as a result of bacterial decarboxylation as fish

samples become stale. However, the observation that

digestion with some peptidases did not inhibit would sug-

gest that any amines generated in this way were not suf-

ficient to inhibit the enzyme.

Further analytical studies should be undertaken to

establish the nature of any inhibitory peptides present in

these hydrolysates as lead compounds for further devel-

opment as PrAO inhibitors.

Acknowledgments The authors wish to thank the Ashtown Food

Research Centre (AFRC), Dublin, Ireland for the provision of some of

the materials used in these studies and the Department of Agriculture,

Fisheries & Food (Ireland) for financial support towards part of this

work.

Conflict of interest The authors declare that they have no conflict

of interest.

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