Variations in the activity of digestive enzymes alongthe intestine of the burbot Lota lota expressedby different methods

Galina I. Izvekova • Mikhail M. Solovyev •

Elena N. Kashinskaya • Evgeny I. Izvekov

Received: 10 October 2012 / Accepted: 2 February 2013 / Published online: 14 February 2013

� Springer Science+Business Media Dordrecht 2013

Abstract The activities of major digestive hydro-

lases (proteases, amylase, lipase and esterases) along

the intestine were studied in the burbot Lota lota (L.)

using different methods of activity expression. The

enzyme activities were determined both in the whole

gut segments and in the isolated mucosa, and then

expressed in terms of tissue mass and protein content

in the samples. Further, the cumulative activities of

these enzymes in the pyloric caeca were compared

with those in the rest of the intestine to estimate the

overall contribution of these regions to digestion. The

data obtained suggest the essential role of the pyloric

caeca in the digestion of the burbot. In addition, the

variations in the pH values along the intestine and the

changes in the enzyme activities with incubation

temperature were examined. The study proved the

method of enzyme activity expression to be a key

factor influencing the outcome of the experiment.

Keywords Fish � Lota lota � Digestion � Enzyme

activity � pH � Temperature

Introduction

The characteristic feature of digestive hydrolases in

fish is their adaptive flexibility related to variable

enzyme distribution in the wall and along the lumen of

the alimentary tract. As a rule, localization and activity

of these enzymes reflect the feeding habits and

intestinal morphology of the fish (Kuz’mina 2008).

Both proximo-distal and radial activity gradients of

various hydrolases are currently known (Chakrabarti

et al. 1995; Lundstedt et al. 2004). Extensive infor-

mation on the activity distribution of various digestive

enzymes along the intestines of different fish species is

inconsistent (Kuz’mina 1979; Deguara et al. 2003;

Lundstedt et al. 2004; Sklan et al. 2004). Nonetheless,

some common patterns in the existence of such

gradients were reported. For example, membrane

hydrolysis of fats occurs mainly in the proximal

section of the intestine and in the pyloric caeca, while

the carbohydrates and proteins are hydrolyzed in the

middle and distal gut sections (Kuz’mina 2008).

The functional topography of fish intestine, even

within a rather uniform group, varies widely, up to the

full reversal of the gradient (Kuz’mina 1979). For

instance, the diet composition plays a significant role in

the formation of proximo-distal gradients along the

intestine (Lundstedt et al. 2004; Sklan et al. 2004). The

studies also showed a possibility of fast gradient

rearrangement in developing animals (Kuz’mina 1996).

The burbot Lota lota (L.) is the only freshwater

member of family Lotidae, with other species inhabiting

G. I. Izvekova (&) � E. I. Izvekov

Papanin Institute for Biology of Inland Waters, Russian

Academy of Sciences, Borok, Nekouzskii raion,

Yaroslavskaya oblast, Moscow 152742, Russia

e-mail: izvekov@ibiw.yaroslavl.ru

M. M. Solovyev � E. N. Kashinskaya

Institute of Systematics and Ecology of Animals, Siberian

Branch of Russian Academy of Sciences, 11 Frunze St.,

Novosibirsk 630091, Russia

123

Fish Physiol Biochem (2013) 39:1181–1193

DOI 10.1007/s10695-013-9773-y

cold marine waters. The burbot experiences consider-

ably larger temperature changes as compared to its

marine ancestors and is well adapted to freshwater

habitats (Hardewig et al. 2004). For instance, in the

Rybinsk reservoir (Russian Federation, 58�2203000N38�2800400E), the burbot spawns and actively feeds in

January and February (Kuperman and Kuz’mina 1994).

Adult burbots are categorized as primary piscivores and

facultative benthivores. The burbot is currently regarded

as a promising finfish species for cold-water aquacul-

ture. The above explains a considerable interest in the

studies on feeding and digestion of the burbot.

Enzyme activity can be expressed by different

methods, and in many cases, the results and their

interpretation would depend on the way of activity

calculation. Most commonly, enzyme activity is given

per gram of intestine tissue or per milligram of protein

contained in a sample. Furthermore, activity can be

determined either in the intestinal mucosa alone

(Kuz’mina 1979) or in the whole gut segment (Chak-

rabarti et al. 1995; Deguara et al. 2003; Day et al. 2011).

The main objective of the study was to examine the

activity distributions for major digestive enzymes

along the burbot intestine, and compare the distribu-

tion patterns using different methods of activity

expression. Besides, the pH values were determined

in different segments of the gut, and the enzyme

activities in these segments measured at various

incubation temperatures as the burbot is a cold-water

species that actively feeds in winter.

Materials and methods

Study specimens and tissue preparations

The study was conducted with adult burbots caught in

the Rybinsk reservoir in January and February 2011 by

local fishermen. The standard length of the fish ranged

from 380 to 460 mm. Fish were taken from the daily gill

net catches, killed by a blow on their head and dissected.

Shortly after capture, the digestive tracts of five

specimens were extracted and split into five segments

(the stomach, pyloric caeca, anterior, middle and

posterior sections). Immediately after dissection, the

pH values were measured in each segment using the

pH meter HI 8314 (Hanna Instruments, Romania) with

microelectrode HI 1083 B (3 mm in diameter).

For enzyme assays, the intestines of 26 burbots

were frozen and stored in liquid nitrogen until

analysis. Before the assays, the intestines were

dissected, the chyme was removed, and each gut was

split into six segments (I pyloric caeca, II–VI further

intestinal segments, each from 4 to 5 cm long).

To compare various methods of activity expression,

the homogenates for enzyme assays were prepared in

two different ways. In the first group of fish (n = 9),

homogenates were obtained from the whole intestinal

segments, while in the second group (n = 17), only

the isolated mucosa of each segment was used. For this

purpose, the intestines were cut lengthwise, cleaned of

chyme, and the mucosa was removed with a plastic

spatula. When preparing the homogenates, 0.1 M

Tris–HCl buffer (pH 7.5) was added to weighed

samples of the gut segments or net mucosa, and the

mixture was centrifuged at 10,000g for 10 min at 4 �C

using the microcentrifuge 5417R (Eppendorf, Ger-

many). The preparation of active enzyme samples was

carried out in an ice bath (at 0 �C).

Enzyme assays

The enzyme activities were determined at ambient

temperature of 25 �C. The total activity of proteinases

(activities of trypsin EC 3.4.21.4, chemotrypsin EC

3.4.21.1, and dipeptidases EC 3.4.13.18) was deter-

mined using 0.3 % (w/v) azocasein as substrate

(Alarcon et al. 2002). The activity of amylase (EC

3.2.1.1) was determined using 1 % (w/v) soluble

starch as the substrate (Deguara et al. 2003). The

activity of lipase (nonspecific lipases EC 3.1.1) was

analyzed with 0.4 mM p-nitrophenyl-myristate as the

substrate (Gawlicka et al. 2000). The activity of

esterases (nonspecific esterases EC 3.1.1) was deter-

mined with 0.27 mM p-nitrophenil acetate as the

substrate (Prabhakaran and Kamble 1995). The sub-

strates for the determination of the total proteolytic

activity and the activity of a-amylase were prepared

using Tris–HCl buffer (pH 7.5); for the determination

of the activities of nonspecific esterases, using 0.1 M

phosphate buffer (pH 7.5); for the determination of

lipase, using 24 mM ammonium bicarbonate (pH 7.5)

with 0.5 % (w/v) triton X-100 added as the detergent.

The dissolved protein concentration in the samples

was measured using the Bradford’s method (Bradford

1976). The color development proportional to enzy-

matic activities was measured using the microplate

1182 Fish Physiol Biochem (2013) 39:1181–1193

123

spectrophotometer Multiskan Ascent (Thermo,

Germany).

Expression of enzyme activities

The activities of the studied enzymes were expressed

as the difference of spectrophotometer readings for the

sample with the substrate versus a blank sample per

minute (DAbs min-1). In four different estimates, this

value was divided by: (1) mass of mucosa in a gut

segment, g; (2) protein content in a mucosa sample,

mg; (3) mass of a gut segment, g; (4) protein content in

a gut segment, mg. In order to evaluate the total

digestive capacities of pyloric caeca and the rest of the

gut, the cumulative activities of each enzyme in these

two regions were calculated. For this purpose, the

activity for each section was multiplied by the

corresponding mass of the gut tissue, with subsequent

summing of the values for all sections. The cumulative

activities for both pyloric caeca and the entire gut were

calculated in two modifications: per total weight of

wet intestinal tissue and per weight of mucosa alone.

Measuring the effects of pH and temperature

on enzyme activities

In a special series of experiments, the proteolytic

activity in the burbot stomach (n = 26) was deter-

mined at two different pH values (3 and 6) with 0.3 %

(w/v) hemoglobin as the substrate (Anson 1938).

Additionally, the activity of each enzyme in all the

intestinal segments of five burbots was measured at

various incubation temperatures (4, 15 and 25 �C),

and averaged for all segments.

Statistical analysis

The results are given as means ± SE. Statistical

analyses were carried out using the software package

STATISTICA 6.0 (StatSoft, Inc., Tulsa, OK, USA).

First, the distribution of each data set was tested for

normality using the Shapiro–Wilk W test. In some

cases, the distributions were close to normal, while in

others, significant departures from normality were

observed. Therefore, nonparametric statistical analy-

ses were applied throughout. Friedman ANOVA with

a posteriori testing was used to examine statistical

differences between the intestinal segments. The post

hoc analyses were conducted using the Wilcoxon

pairwise t test with Bonferroni adjustment for multiple

comparisons. When comparing the mucosa and seg-

ment samples within any particular portion of the gut,

the Mann–Whitney U test was used. All the tests were

two-tailed, and the level of significant difference was

set at P \ 0.05.

Results

Variations in pH values and protein content

along the digestive tract

The lowest pH values (5.08 ± 0.43 as average) were

found in the burbot stomach (Fig. 1). These values

were significantly lower compared with those for

pyloric caeca or any intestinal segment (P \ 0.05)

which were superior to 7. The pH reached the

maximum values in the anterior section of the intestine

and decreased toward the posterior segment.

The greatest protein content was detected in the

pyloric caeca compared to the intestine, both in the

whole segments and in the mucosa (P \ 0.01), and in

most cases, concentrations of protein in the mucosa

were higher than in the whole segments, with signif-

icant differences (P \ 0.01) for pyloric caeca

(Table 1).

Enzyme activities expressed per tissue mass

and protein content

The distributions of enzyme activities along the

intestinal tract of the burbot are given in Figs. 2 and

3. As a rule, the proteolytic activity in the pyloric

caeca was lower compared to the rest of the intestine,

bdbc

c

b

a

4

5

6

7

8

S PC A M P

Sections of digestive tract

Fig. 1 The pH values (mean ± SE, n = 5) in different sections

of the gastrointestinal tract of the burbot: S stomach, PC pyloric

caeca, A anterior, M middle, P posterior. Means with different

letters are significantly different (P \ 0.05)

Fish Physiol Biochem (2013) 39:1181–1193 1183

123

whatever the way of representation (Fig. 2a, b). In

case of mucosa samples, significant (P \ 0.05) dif-

ferences were observed between the pyloric caeca and

the next gut segment (II). At the same time, no

significant differences in proteinase activities were

found among the intestinal segments (II–VI), irre-

spective of the calculation method.

The calculation method considerably influenced the

pattern of activity distribution along the gut for

amylase, lipase and esterases (Figs. 2c, d, 3a–d). For

Table 1 Concentrations of protein (mg ml-1) in the intestine and pyloric caeca of the burbot

Samples Segments

I II III IV V VI

Segment (n = 9) 1.55* ± 0.28a 0.86 ± 0.20b 0.67 ± 0.15c 0.53 ± 0.06c 0.80 ± 0.25bc 1.29 ± 0.22ab

Mucosa (n = 17) 4.21* ± 0.49a 0.96 ± 0.11b 0.99 ± 0.21b 1.11 ± 0.30b 1.37 ± 0.30b 1.16 ± 0.22b

Each value represents mean ± SE. I pyloric caeca, II–VI further intestinal segments, n fish number

Different superscripts show significant differences between the data in a row (i.e., between the different gut segments) (P \ 0.01).

Asterisks show significant differences between the data in a column (i.e., between the segment and mucosa samples) (P \ 0.01)

b

aa

aa

aa

AA

A

AA

A

0.0

0.2

0.4

0.6

I II III IV V VI

Abs

min

–1 g

–1 s

egm

ent

0.00

0.02

0.04

0.06

Abs

min

–1 m

g–1

pro

tein

3 4

ab

ababab

b

a

A

A

A

AA

A

0.00

0.01

0.02

0.03

0.04

I II III IV V VI

Abs

min

–1 g

–1 s

egm

ent

0.000

0.001

0.002

0.003

0.004

Abs

min

–1 m

g–1

pro

tein

d

a

abab

abab

b

a

ABAB

AB

AB

B

A0.0

0.1

0.2

0.3

0.4

0.5

I II III IV V VI

Abs

min

–1 g

–1 m

ucos

a

0.00

0.01

0.02

0.03

Abs

min

–1 m

g–1

pro

tein

1 2

aa

aa

a a

A

B BB

AB AB

0.00

0.02

0.04

0.06

I II III IV V VI

Abs

min

–1 g

–1 m

ucos

a

0.000

0.001

0.002

0.003

0.004

Abs

min

–1 m

g–1

pro

tein

c

Protease activity (mucosa)

Amylase activity (segment)

Protease activity (segment)

Amylase activity(mucosa)

Fig. 2 Proteolytic (a, b) and amylolytic (c, d) activities in

different segments of the burbot intestine. 1 DAbs min-1 g-1

mucosa, 2 DAbs min-1 mg-1 protein in mucosa, 3 DAbs

min-1 g-1 segment, 4 DAbs min-1 mg-1 protein in segment, I

pyloric caeca, II–VI further intestinal segments. Mean ± SE

indicated, n = 17 (a, c), n = 9 (b, d). Within the same curve,

values marked with different letters are significantly different

(P \ 0.05)

1184 Fish Physiol Biochem (2013) 39:1181–1193

123

instance, when the amylase activity was expressed per

gram of mucosa or per milligram of protein in the

whole segments, no clear distribution pattern was

seen. On the contrary, when using two other methods

of activity expression, there were significant differ-

ences (P \ 0.05) between the amylase activity levels

in the pyloric caeca and those in the next gut segments.

When expressing the results per tissue mass unit,

the activities of lipase and esterases in the pyloric

caeca were significantly (P \ 0.05) higher than those

in the next gut segments (both in the mucosa samples

and in the whole gut segments). However, when these

activities were given per milligram of protein, this

difference reversed its sign (in the mucosa samples), or

tended to disappear (in the gut segments) (Fig. 3).

The activities of amylase, lipase and esterases per

gram of tissue were significantly higher for the mucosa

samples than for the whole gut segments in most

intestinal sections (Table 2). On the other hand, in

terms of protein, no significant differences in amylase

activity were found between mucosa and segment

samples, while in case of lipase and esterases, the

lower activity values were observed in mucosa

(Table 2).

In addition, the A/P ratio (i.e., amylase activity

divided by the activity of proteases) was calculated for

each segment (Table 3). This ratio was less than 1 in

each segment of the burbot intestine. When expressing

enzyme activity per gram of mucosa, the A/P ratio for

pyloric caeca was 2–2.4 times higher than that for the

d

Ab b b b

A

a A

A A

A b

0

1

2

3

4

I II III IV V VI

Abs

min

–1 g

–1 s

egm

ent

0

0.1

0.2

0.3

Abs

min

–1 m

g–1

pro

tein

b

A

A

AAA

A

b

bbb

b

a

0.0

0.2

0.4

0.6

I II III IV V VI

Abs

min

–1 g

–1 s

egm

ent

0.00

0.01

0.02

0.03

0.04

0.05

Abs

min

–1 m

g–1

pro

tein

3 4

c

a

bb ab

ab ab

A

B

B

ABAB AB

0

1

2

3

4

I II III IV V VI

Abs

min

–1 g

–1 m

ucos

a

0.0

0.1

0.2

0.3

Abs

min

–1 m

g–1

pro

tein

a

a

b

acac

ac ac

A

B B B

AB AB

0.0

0.2

0.4

0.6

I II III IV V VI

Abs

min

–1 g

–1 m

ucos

a

0.00

0.01

0.02

0.03

Abs

min

–1 m

g–1

pro

tein

1 2

Esterase activity (mucosa)

Lipase activity (mucosa)

Esterase activity (segment)

Lipase activity (segment)

Fig. 3 Lipase (a, b) and esterase (c, d) activities in different segments of the burbot intestine. See Fig. 2 for elucidation

Fish Physiol Biochem (2013) 39:1181–1193 1185

123

rest of the gut, while per milligram of protein, smaller

differences (1.2–1.6 times) were observed.

Cumulative enzyme activities in different parts

of the gut

Along with the above calculations per mass unit, the

cumulative activities of the enzymes localized in the

pyloric caeca or immediately in the intestine were

determined (Table 4). When considering the overall

mass of mucosa, the cumulative proteolytic activities

in the intestine were significantly greater than those in

the pyloric caeca, while the rest of the enzymes

displayed no significant localization differences. On

the contrary, the cumulative activities of proteinases in

the gut and pyloric caeca were similar, with signifi-

cantly higher activity of the other enzymes found in

the caeca. In the pyloric caeca, the total mass of

mucosa was considerably greater than in the gut

(P \ 0.01), while the total masses of all caeca and the

whole intestine were comparable (Table 5).

The calculations of enzyme activity per total

protein content in the sample showed that in terms of

net mucosa, the total activities of proteinases or lipase

in the pyloric caeca were significantly higher as

compared with the intestine, and the opposite was true

for amylase and esterase. At the same time, when the

whole tissue segments were used, no significant

differences in total enzyme activities between the

pyloric caeca and the intestine were found (Table 4).

Both for mucosa and for gut segments, the total protein

content in the pyloric caeca was significantly higher

than that in the intestine (Table 5). The cumulative

enzyme activities calculated per total mass of seg-

ments were higher than those per total mass of mucosa

both for pyloric caeca and for the entire gut, and most

differences were significant (P \ 0.05) (Table 4).

pH and thermal dependence of enzyme activities

In a special series of experiments conducted at two

different pH values, proteolytic enzymes in the

stomach were active at both pHs 3 and 6. However,

at pH 3, proteinase activity was almost 4 times higher

than at pH 6 (0.395 ± 0.045 and 0.099 ± 0.015 DAbs

min-1 mg-1 protein, respectively; P \ 0.01).

Additionally, the activities of major digestive

enzymes of the burbot were studied at various

temperatures of the incubation media (Fig. 4). Activ-

ities of all studied enzymes both in the pyloric caeca

and in the intestine tended to rise as incubation

temperature increased from 4 to 25 �C. The largest

increase was noted for proteinases (89 % in the

temperature range from 4 to 25 �C). Within the same

temperature interval, amylase activity increased by

46–51 %, and the activities of lipase and esterases

raised only by 28–31 % (Fig. 5). In most cases, the

activities of the same enzyme in the pyloric caeca and

Table 2 Comparison of digestive enzyme activities in the mucosa (M, n = 17) and gut segments (S, n = 9) of the burbot expressed

in DAbs min-1 g-1 tissue (a) and DAbs min-1 mg-1 protein (b) (Mann–Whitney U test)

Gut segment Proteinases Amylase Lipase Esterases

a

M \ S

b

M \ S

a

M [ S

b

M \ S

a

M [ S

b

M \ S

a

M [ S

b

M \ S

I P \ 0.05 P \ 0.05 P \ 0.05 P \ 0.1 – P \ 0.05 – P \ 0.05

II – P \ 0.05 P \ 0.05 – – P \ 0.05 P \ 0.1 P \ 0.1

III – P \ 0.05 P \ 0.05 – P \ 0.1 P \ 0.05 P \ 0.05 –

IV – P \ 0.05 P \ 0.05 – – P \ 0.05 P \ 0.1 P \ 0.1

V – P \ 0.1 P \ 0.05 – P \ 0.05 P \ 0.05 P \ 0.05 P \ 0.1

VI – – P \ 0.05 – P \ 0.05 – – P \ 0.05

Dashes denote the absence of significant differences

Table 3 Variation in A/P ratio values along the intestine of

the burbot

Calculation method Segments

I II III IV V VI

Per gram of mucosa 0.39 0.16 0.19 0.17 0.20 0.16

Per milligram of

protein

0.22 0.14 0.18 0.13 0.14 0.13

I pyloric caeca, II–VI further intestinal segments

1186 Fish Physiol Biochem (2013) 39:1181–1193

123

in the intestine grew proportionally as temperature

increased. The distribution of the studied enzymes

along the intestine did not depend on the temperature

of the incubation medium.

Discussion

Changes in pH and protein content

along the alimentary canal

Changes in pH values along the gastrointestinal tract

are known for all vertebrates. The stomach is the

digestive organ where enzymatic reactions occur in

the acid medium. The pH values in the stomach of the

burbot determined in our experiments were somewhat

higher compared to those for other fish species which

usually range from 2.88 to 4.25 (Clements and

Raubenheimer 2005). At the same time, the pH values

in the burbot intestine varied within the limits reported

for other fishes (from 6.5 to 7.9) (Deguara et al. 2003;

Bakke et al. 2010).

The uneven distribution of the protein content per

gram of mucosa along the intestine found in our study

apparently reflects the morphological differences

between the gut regions. The mucosa of actively

feeding fish is lined with single-layer cylindrical

epithelium bearing the brush border that consists of

numerous microvilli (Kuperman and Kuz’mina 1994).

The intestinal epithelium is composed of columnar

cells (enterocytes) and goblet (mucous) cells located

nonuniformly along the intestine (Kuz’mina 1995).

Goblet cells secret a mucous fluid containing high-

molecular proteids. Microvilli are also covered with a

glycocalyx layer formed by oligosaccharide chains of

glycoproteids and glycolipids of the plasmalemma

Table 4 Cumulative activities of digestive enzymes in the pyloric caeca (PC) and intestine (I) of the burbot expressed in different

ways

Enzymes Cumulative activity

PC1 I1 PC2 I2

Per total mass of mucosa (n = 17) Per total mass of gut tissue (n = 9)

Proteases 0.197 ± 0.049a,b 0.563 ± 0.076a,b 1.882 ± 0.329b 2.621 ± 0.751b

Amylase 0.091 ± 0.016b 0.101 ± 0.012 0.178 ± 0.017a,b 0.129 ± 0.015a

Lipase 0.987 ± 0.154b 0.874 ± 0.098b 2.816 ± 0.259a,b 2.079 ± 0.378a,b

Esterases 5.724 ± 0.809b 5.486 ± 0.592b 16.539 ± 1.742a,b 10.140 ± 0.999a,b

Per total amount of protein in mucosa (n = 17) Per total amount of protein in gut tissue (n = 9)

Proteases 0.093 ± 0.028 0.030 ± 0.005b 0.141 ± 0.034 0.266 ± 0.082b

Amylase 0.002 ± 0.000a,b 0.005 ± 0.001a,b 0.009 ± 0.001b 0.013 ± 0.002b

Lipase 0.224 ± 0.034a 0.037 ± 0.005a,b 0.187 ± 0.030 0.169 ± 0.030b

Esterases 0.137 ± 0.020a,b 0.255 ± 0.041a,b 0.917 ± 0.098b 1.146 ± 0.242b

a Denotes significant differences (P \ 0.05) between PC1 and I1, or between PC2 and I2

b Denotes significant differences (P \ 0.05) between PC1 and PC2, or between I1 and I2

Values are mean ± SE

Table 5 Total mass (g) of tissue samples and total protein content (mg) in different parts of the burbot intestine

Mucosa (n = 17) Gut segment (n = 9)

Pyloric caeca Intestine Pyloric caeca Intestine

Tissue mass

3.54 ± 0.32 2.04 ± 0.12 6.35 ± 0.53 5.86 ± 0.37

Protein

16.18 ± 2.87 2.52 ± 0.53 4.68 ± 0.67 1.56 ± 0.20

Each value represents mean ± SE

Fish Physiol Biochem (2013) 39:1181–1193 1187

123

a

a b bA

B

C

0.000

0.004

0.008

0.012

0.016

0.020

4° 15° 25°

Abs

min

-1 m

g-1 p

rote

in

Pyloric caeca

Intestine

b

ab

c

A

B

C

0.000

0.001

0.002

0.003

0.004

4° 15° 25°

c

aa

bA

A

B

0.000

0.005

0.010

0.015

0.020

0.025

4° 15° 25°

Temperature

Abs

-1m

g-1 p

rote

in

d

a a

b

AA

B

0.00

0.04

0.08

0.12

0.16

4° 15° 25°

Temperature

Proteinases Amylase

Lipase Esterases

Fig. 4 Mean ± SE activities of digestive enzymes in burbot at different incubation temperatures. Bars with different lower case letters

(pyloric caeca) or different upper case letters (intestine) are significantly different (P \ 0.05, n = 5)

-20

0

20

40

60

80

100

PP I PP I PP I PP I

Proteinases Amilase Lipase Esterases

4–15°C15–25°C

4–25°C

Act

ivity

cha

nges

(%

)

Fig. 5 Relative changes (%) in the activities of burbot digestive enzymes in response to elevation of the incubation temperature. PP

pyloric caeca, I intestine. Mean ± SE indicated (n = 5)

1188 Fish Physiol Biochem (2013) 39:1181–1193

123

(Kuz’mina 1995). The ultrastructure analysis of the

enterocytes from different parts of the intestine shows

considerable differences in the structural organization

of the brush border in the burbot. In mid-intestine, the

brush border bears longer and more widely spaced

microvilli compared to those in the anterior part, in the

region of pyloric caeca (Kuperman and Kuz’mina

1994). Our data on the protein content in the mucosa of

the burbot agree with the nonuniform distribution of

goblet cells and glycocalyx layer along the fish

intestine.

When measuring the enzyme activity in the intes-

tine, the weighed sample, besides mucosa, contains

submucous, muscular and serous layers. The higher

protein contents per gram of mucosa relative to those

for the gut segments could be explained by the fact that

the method used allows determining only soluble

protein in a sample (Bradford 1976). In turn, the

concentration of soluble protein in mucosa would be

higher than in the muscular and serous layers that both

contain a greater portion of insoluble structural

proteins. These differences in protein content could

affect the activity levels of the studied enzymes.

Enzyme activity patterns along the gut depending

on the method of activity expression

Differences in activity of digestive enzymes depend-

ing on the method of its expression were also noted by

other authors (Kuz’mina et al. 1982; Chan et al. 2004).

Expressing the activity per mass of intestinal tissue or

per protein mass can change the absolute activity

values, and even cause the reversal of inter-species

differences (Chan et al. 2004), which is probably

associated with differences in protein content between

the samples.

Similar levels of proteolytic activity in the mucosa

and intestine segments calculated per gram of tissue

(Table 2) suggest rather uniform distribution of pro-

teinases between the mucosa and intestinal wall. These

data agree with the earlier findings, showing a

considerable proteolytic activity in the muscular layer

of the intestine in the bream Abramis brama (Ugolev

and Kuz’mina 1992). The comparative analysis of

amylase, lipase and esterase activities per 1-g mucosal

and intestinal weighed samples in the burbot has

shown these enzymes to be localized mostly in the

mucosa. In the burbot, lower levels of protease, lipase

and esterase activities in mucosa versus segment

samples when expressing activity in milligrams of

protein could be explained by higher protein content in

the mucosa. The same explanation would be reason-

able for the insignificant differences between the

amylase activities in the mucosa and whole segments.

Terrestrial vertebrates generally display relative

functional specialization of different sections of their

small intestine. The distribution of enzyme and

transport activities along the small intestine is related

to the adaptation of its parts to various functional loads

(e.g., changes in diet composition, velocity of chyme

movement) (Ugolev and Iezuitova 1985). In fishes, the

activities of major digestive enzymes (trypsin, amy-

lase, lipase, etc.) are present along the whole length of

the digestive tract (Tengjaroenkul et al. 2000; Deguara

et al. 2003; Lundstedt et al. 2004; Sklan et al. 2004). In

our experiments, the activities of proteinases, amylase,

lipase and esterases were also found all along the

burbot intestine.

The lack of clear gradients in the enzyme distribu-

tion within sections II–VI of the burbot intestine

corresponds to the data by other authors (Kuz’mina

1979; Deguara et al. 2003; Lundstedt et al. 2004; Day

et al. 2011). They have shown that in many fish

species, the proximo-distal gradients of various

enzymes (trypsin, chymotrypsin, aminopeptidase,

amylase, maltase and lipase) could be feebly marked.

The A/P ratio is used to evaluate the relative

activities of amylolytic and proteolytic enzymes in the

intestine of fish. This ratio reflects the feeding habits of

the fish: in predators, it is less than 1, whereas in

planktophages and benthophages, it is usually more

than 1 (Ugolev and Kuz’mina 1994; Kuz’mina 1996).

The A/P values obtained for the burbot agree with this

concept. In addition, the A/P ratio in the intestine

decreases caudally, suggesting the greater contribu-

tion of proteases to digestion in the burbot intestine

compared with the pyloric caeca.

The activities of all tested enzymes in the pyloric

caeca converted to DAbs min-1 per milligram of

protein were lower than those in the other parts of the

gut. At the same time, the activities of lipase and

esterases calculated per gram of tissue in the pyloric

caeca exceeded those in further segments, which is

consistent with the concept of pyloric caeca signifi-

cance for hydrolysis of fats (Kapoor et al. 1975; Fange

and Grove 1979). The lower levels of enzyme activity

in the pyloric caeca are apparently related to the higher

protein content per gram of mucosa in this region of

Fish Physiol Biochem (2013) 39:1181–1193 1189

123

Ta

ble

6E

xam

ple

so

fen

zym

eac

tiv

ity

dis

trib

uti

on

alo

ng

the

dig

esti

ve

trac

tin

fish

es

Fis

hsp

ecie

sE

nzy

mes

Act

ivit

yd

istr

ibu

tio

nR

efer

ence

s

Nil

eti

lap

iaO

reo

chro

mis

nil

oti

cus

(om

niv

oro

us,

mo

stly

her

biv

oro

us)

Mal

tase

Am

ino

pep

tid

ase

and

alk

alin

e

ph

osp

hat

ase

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ase

No

nsp

ecifi

ces

tera

ses

Seg

men

t3

ou

to

f5

(max

imu

m)

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men

ts1

–3

ou

to

f5

(max

imu

m)

Seg

men

ts1

and

2o

ut

of

5(m

axim

um

)

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men

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–4

ou

to

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(max

imu

m)

Ten

gja

roen

ku

l

etal

.(2

00

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bri

dti

lap

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.n

ilo

ticu

s9

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au

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ryp

sin

,am

yla

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dli

pas

eD

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ases

dis

tall

yS

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net

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(20

04

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thea

dse

ab

ream

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aru

sa

ura

ta(o

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my

lase

,tr

yp

sin

and

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mo

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psi

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bo

xy

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dB

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end

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n

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reas

esd

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uar

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(20

03

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pC

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nu

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rpio

(om

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egm

ents

2an

d4

ou

to

f5

(max

imu

m)

Jon

aset

al.

(19

83

)

Sil

ver

carp

Hyp

op

hth

alm

ich

thys

mo

litr

ix(h

erb

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rote

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(19

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)

Eas

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riv

erg

arfi

shH

ypo

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hu

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gu

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sa

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(her

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yla

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try

psi

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op

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das

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d

lip

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end

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(max

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Day

etal

.(2

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1)

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uth

east

ern

snu

b-n

ose

dg

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am

ph

us

scle

role

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(her

biv

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yla

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mal

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,tr

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,

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e

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end

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nD

ayet

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(20

11)

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ng

tom

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on

gyl

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(car

niv

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us)

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yla

se

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n

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ino

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tid

ase

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tase

and

lip

ase

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men

t1

ou

to

f4

(max

imu

m)

Seg

men

t3

ou

to

f4

(max

imu

m)

Seg

men

ts2

and

3o

ut

of

5(m

axim

um

)

Ev

end

istr

ibu

tio

n

Day

etal

.(2

01

1)

Sto

ut

lon

gto

mT

ylo

suru

sg

avi

alo

ides

(car

niv

oro

us)

Am

yla

se

Mal

tase

Try

psi

nan

dam

ino

pep

tid

ase

Lip

ase

Seg

men

t2

ou

to

f4

(max

imu

m)

Seg

men

ts1

–3

ou

to

f4

(max

imu

m)

Ev

end

istr

ibu

tio

n

Seg

men

t3

ou

to

f4

(max

imu

m)

Day

etal

.(2

01

1)

Sp

ott

edso

rub

im(‘

pin

tad

o’

catfi

sh)

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ud

op

laty

sto

ma

orr

usc

an

s(c

arn

ivo

rou

s)

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yla

se

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psi

n

Lip

ase

Max

imu

mac

tiv

ity

inth

est

om

ach

or

even

dis

trib

uti

on

(dep

end

ing

on

die

t)

Sto

mac

h(m

axim

um

)

Seg

men

t2

ou

to

f3

(max

imu

m)

Lu

nd

sted

tet

al.

(20

04

)

Sh

eatfi

shS

ilu

rus

gla

nis

(car

niv

oro

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Pro

teas

esIn

crea

ses

dis

tall

yJo

nas

etal

.(1

98

3)

1190 Fish Physiol Biochem (2013) 39:1181–1193

123

digestive tract. Thus, the uneven distribution of

protein along the intestine affects the profiles of

enzyme activity expressed in DAbs min-1 per milli-

gram of protein.

Currently, there is no common opinion on the

functions of pyloric caeca and relation of their

location, number and structure with feeding habits of

fish. In some species, the most intensive hydrolysis of

lipids was shown to occur in the proximal sections of

the gut (Kapoor et al. 1975; Fange and Grove 1979),

though the other intestinal regions can also participate

in digestion of fats (Chakrabarti et al. 1995; Day et al.

2011). Moreover, noticeable activities of maltase,

trehalase, dextrin-a-(1–6) glucosidase and amylase

were present in the pyloric caeca of the milkfish

Chanos chanos (Chiu and Benitez 1981). According to

our data, the pyloric caeca show similar or even higher

cumulative activities of the studied enzymes com-

pared to those found in the intestine. The higher

cumulative activity of enzymes per segment mass

could be explained by the greater mass of segments

(both for pyloric caeca and for gut fragments) relative

to the mucosa mass in these intestinal regions. These

data suggest the considerable contribution of the

pyloric caeca to the digestion of the burbot.

pH and temperature dependence of enzyme

activities

It is known that both in the stomach and in the intestine

of fishes, the pH values are generally outside the

optima cited for a number of digestive enzymes (Chiu

and Benitez 1981; Deguara et al. 2003). These findings

accord with our data on the burbot proteolytic activity:

stomach proteinases displayed higher activity at pH 3,

while the actual pH values in this part of the digestive

tract averaged 5.08 ± 0.43. As it was shown by other

authors (Sabapathy and Teo 1993; Deguara et al.

2003), the pH values in the intestine are significantly

different from those in the stomach. In some fishes,

pepsin activity is found exclusively in the stomach

(Deguara et al. 2003). However, according to other

reports, a fairly high pepsin activity is also present in

the intestine (Sabapathy and Teo 1993). Besides, the

pH values have a greater effect on the pepsin activity

that drops to zero at pH 5. At the same time, the

trypsin-like enzymes with pH optima between 7 and 8

remain active between 3 and 5 (de la Parra et al. 2007).

The above explains why the proteolytic activity in the

burbot’s stomach is manifested at both pH values 3

and 6.

Our research also showed that all the enzyme

activities tested both in the gut and in the pyloric caeca

grew as the temperature increased. A rise in digestive

enzyme activity with temperature was noted by many

authors (Chiu and Benitez 1981; Jonas et al. 1983;

Kuz’mina 2001). Our data suggest a particular sensi-

tivity of intestinal proteinases in the burbot to an

increase in temperature compared to the other

enzymes.

Distribution of enzyme activity along the intestine

in various fish species

The available data testify to a great diversity in

distribution of enzyme activities along the digestive

tract in fishes with different feeding preferences

(Table 6). Along with the above species differences,

the activity of digestive hydrolases reveals consider-

able individual variability, including both activity

levels and distribution patterns of the same enzymes

(Ugolev and Kuz’mina 1994; Kuz’mina 2001). The

lack of correspondence between the available data on

the distribution of enzyme activity along the fish gut

can have multiple causes, such as differences in life

patterns of the fish, their feeding habits and diet, age,

physiological state and health of the individuals,

season of investigation, arbitrary segmentation of the

gut, different methods of enzyme assay, and other

factors hard to identify. Moreover, it was shown that

the activity pattern is essentially influenced by the way

of activity expression (e.g., either in terms of mucosa

mass unit or in terms of length and surface area of the

intestine) (Kuz’mina et al. 1982), which is also

supported by the results of the present study.

Our data on the activity distributions of digestive

enzymes in the intestine of the burbot, along with some

other reports (Tengjaroenkul et al. 2000; Deguara et al.

2003; Lundstedt et al. 2004; Sklan et al. 2004), confirm

the well-established concepts on the functioning of

digestive hydrolases in fish (Kuz’mina 2001). Taken

together, these findings show that owing to its

extraordinary plasticity, inclusive of variable func-

tional topography, the enzyme system of the intestinal

mucosa plays a significant role in effective nutrition of

fish. The results of these studies also suggest that

various methods for enzyme activity expression could

reflect different aspects of digestion physiology, thus

Fish Physiol Biochem (2013) 39:1181–1193 1191

123

leading to opposite conclusions on the relative signif-

icance of different gut portions in digestion.

Conclusions

Our research has shown considerable variations in pH

values, mucosa mass, content of soluble protein and

activity of digestive enzymes along the gastrointesti-

nal tract of the burbot. The evaluation of the enzyme

activity levels and the determination of activity

distribution along the gut were greatly influenced by

the method of expression of the measured activity.

Based on the A/P ratio values (amylolytic activity

divided by the proteolytic activity), proteinases have a

greater contribution to digestion in the intestine than in

the pyloric caeca. Also, the activity of proteinases

appears to be more temperature-dependent in com-

parison with other enzymes. In the pyloric caeca, the

greater mass of mucosa and higher protein content

compared to those in the intestine were found. As a

consequence, for the majority of calculation methods,

the cumulative activities of each tested enzyme in the

pyloric caeca were comparable to or even higher than

those in the intestine, suggesting the essential role of

the pyloric caeca in the digestion of the burbot.

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