Download Effect of peptide chain length on amino acid and

Clinical Science( 1986) 71,65-69
65
Effect of peptide ch in length on mino acid and nitrogen
absorption from two lactalbumin hydrolysates in the normal
human jejunum
G. K. GRIMBLE, P. P. KEOHANE, B. E. HIGGINS, M. V. KAMINSKI, JR*
AND D. B. A. SILK
Department of Gastroenterologyand Nutrition, Central Middlesex Hospital, London, and * The Chicago Medical
School, Chicago, Illinois, U.S.A.
(Received 6 September 1985/9January 1986; accepted 18 February 1986)
Sumrnary
1. A double lumen jejunal perfusion technique
has been used in man to study the effect of peptide
chain length on absorption of amino acid nitrogen
from two partial enzymic hydrolysates of lactalbumin.
2. Copper-chelation chromatography showed
that one lactalbumin hydrolysate (LH2) contained
98% peptides with a chain length > 4, whilst the
other (LH1) contained a more even spread of chain
lengths with 55% < 4.
3. Absorption of total nitrogen and of 14 amino
acid residues occurred to a significantly greater
extent from the low molecular weight LH1 than
from the higher molecular weight LH2.
4. The results suggest that the pattern of
nitrogen and amino acid absorption from partial
enzymic hydrolysates of whole protein is markedly
influenced by peptide chain length and that brush
border peptide hydrolysis has an important rate
limiting effect on absorption rates.
mammalian small intestine [ 1-31. Similar experiments have shown differences in the kinetics of free
amino acid and peptide transport [4] and many diand tri-peptide bound residues have been shown to
be absorbed faster than when presented in the free
form [l-31. Similar observations were also made
from human intestinal perfusion studies with partial
enzymic hydrolysates of whole protein (consisting
of heterogenous mixtures of small peptides [5-7]),
but there were considerable variations in amino
acid absorption patterns with different hydrolysates
[8].The nature of the starter protein and the type of
hydrolysis used to produce the peptide mixture
have both been shown to influence absorption properties [9]. However, the effect of peptide chain
length on absorption was not studied. The aim of
the present study was therefore to investigate the
influence that peptide chain length has on intestinal
absorption of partial enzymic hydrolysates of lactalbumin in man.
Methods
Key words: amino acid nitrogen absorption, intestinal perfusion, lactalbumin hydrolysate, peptide
absorption, peptide chain length.
Introduction
Recent work has established the existence of at least
one transport system which mediates absorption of
unhydrolysed model di- and tri-peptides from
Correspondence: Dr D. B. A. Silk, Department of Gastroenterology and Nutrition, Central Middlesex Hospital,
Acton Lane, London NWlO 7NS.
Enzymic hydrolysates and amino acid mixtures
Two partial enzymic hydrolysates of lactose-free
lactalbumin (approx. 92% protein) were prepared
from a single protein source. Papain and trypsin
were used to hydrolyse the protein in vitro and the
rate and extent of hydrolysis was measured titrimetrically until the reaction was stopped at the desired
end-point by heating to 100°C. The remaining
enzyme and undigested protein ‘cores’were precipitated by sedimentation and the resulting supernatant was decolorized, using activated charcoal,
passed through a 0.45 p m prefilter and ultrafiltered
66
G. K . Grimble et al.
using a Millipore Pellicon cassette system (type
PTCG with a molecular weight cut-off of 10000)
before freeze-drying. On two separate occasions,
the reaction time was adjusted to produce two
hydrolysates of similar amino acid composition but
of different chain length profile. Full details of the
method of preparation of hydrolysates are the subject of a US. Patent Application (reference no.
27083-00).
TABLE1. Chain length distribution of lactalbumin
hydrolysates
Determined by Cu(I1)-Sephadex chromatography
as described in the text.
Mean chain length
Distribution (“hby wt.)
Chainlength ... > 4
LH1 3.85
LH2 7.7
40
98
4
3
2
1
23
-
22
-
10
-
5
2
Determination of peptide chain length
Peptide chain length was determined by ligand
exchange chromatography using Cu(I1)-Sephadex
as described by Rothenbuhler et al. [lo]. A column
(1.6 cm X 40 cm) was packed with Cu(I1)-Sephadex, equilibrated with 50 mmolA disodium tetraborate (pH 11.0) and the column eluent was
monitored at 254 nm to detect Cu(I1)-peptide complexes. This column was calibrated with synthetic
di- and tri-peptides (dipeptides: Lys -Asp,
Ala - Gly, Pro - Val, Ala - Tyr, Asp - Ala, Arg Tyr, Glu -Val, Ala - His, Gly - Leu, Arg - Arg,
Glu - Glu, Leu - Gly, Met - Ser, Leu - Phe, His Leu; tripeptides: Leu - Ala - Pro, Glu - Thr - Tyr,
Phe - Gly - Gly, Gly - Ala - Gly, Glu - Cys - Gly),
eluted using the same buffer. Approximate retention times were thus obtained for each oligopeptide
chain length.
For analysis of an unknown oligopeptide mixture, 3 mg of peptide was dissolved in 1 ml of buffer
and applied to the column. The mixture was then
fractionated into different class sizes according to
the retention times predetermined above. The precise mean chain length of each fraction was analysed
by determination of a-amino nitrogen, before and
after total acid hydrolysis [ll, 121, a method similar
to that previously used to determine the mean chain
length of hydrolysis products of protein in the rat
jejunum [13]. The mean chain length obtained for
each oligopeptide class varied somewhat with each
separation but was always within the limits: ‘pentapeptides’ 5 f 0.3, ‘tetrapeptides’ 4 k 0.3, ‘tripeptides’ 3 f 0.3, ‘dipeptides’2 f 0.3. The oligopeptide
distribution (by weight) was thus obtained by integration of the total quantity in each size-fraction
(Table l ) , whilst the percentage of free amino acids
was calculated by the difference. The amino acid
composition of each preparation was analysed after
acid hydrolysis, in 4.0 molA methanesulphonic acid
at 110°Cfor 24 h in vacuo, on a Locarte automatic
amino acid analyser with an Apple IIe integration
system (Locarte Company, London) (Table 2).Two
free amino acid mixtures simulating the amino acid
composition, of complete acid hydrolysates, of LH1
and LH2 [5-81 were also prepared, taking into
account losses of threonine and serine on hydroly-
TABLE
2. Amino acid composition of lactalbumin
hydrolysates
Amino acid
Amino acid composition
(residued100)
Low mol. wt.
lactalbumin
hydrolysate
Higher mol. wt.
lactalbumin
hydrolysate
(LH2)
Asx
Thr
Ser
Glx
Pro
GlY
Ala
Val
CYS
Met
Ile
Leu
TYr
Phe
His
TrP
LYS
‘4%
9.73
5.75
3.47
19.47
5.75
5.09
8.99
5.91
1.10
2.38
4.36
12.19
1.43
2.57
2.39
1.60
7.53
0.30
10.52
5.95
5.76
16.78
4.86
3.67
7.67
5.99
1.06
2.07
4.63
12.16
2.86
2.90
2.00
n.d.
9.27
1.84
sis. Since the proportion of asparagine and glutamine could not be determined by this method, the
amino acid mixture contained aspartate and glutamate. The differences in amino acid composition
between hydrolysates are likely to have been caused
by removal of some residues by the precipitation
and decolorization steps.
Perfusion technique
Six normal healthy volunteers were intubated
with a double lumen perfusion tube incorporating a
proximal occlusive balloon [13]. The study was
approved by the Ethical Committee of the Central
Middlesex Hospital. Full details of the perfusion
technique and methods used for collecting intestinal
aspirates have been described in detail [5-7, 141.
Peptide chain length and jejunal absorption
Each subject was perfused in random order with
four test solutions, each containing one of the lactalbumin hydrolysates or their free amino acid mixtures. All test solutions contained 100 mmolA total
amino acid. The test solutions contained 1 pCi (37
kBq) of ''C-PEGA [15] and the tonicity and pH of
the solutions were adjusted to 290-300 mosmol/lcg
and 7.0 by addition of NaCl and NaOH respectively. The estimated irradiation to the gut from
14C-PEGwas 20 mrad.
Analytical methods and calculation of results
The amino acid content of test solutions and
intestinal aspirates was determined by automated
amino acid analysis (Locarte Company, London)
after complete acid hydrolysis with 4.0 moVl
methanesulphonic acid in vacua. Nitrogen was
measured by an automated chemiluminescence
technique [Antek Model 703 nitrogen analyser,
Edect (Scientific) Ltd, Northants., U.K.]. The 14CPEG content of samples was measured by liquid
scintillation counting with quench correction by the
H-number technique (Beckman LD7S00, Beckman-Riic, High Wycombe, Bucks., U.K.). Sodium
content of perfusates and aspirates was measured
by flame photometry. Amino acid absorption was
calculated using previously described formulae [ 161.
Luminal disappearance of amino acid residues was
taken to be equivalent to absorption. The significance of differences was tested by the t-test.
67
Results
Ten amino acid residues (Asx, Thre, Ser, Glx, Ala,
Gly, Val, Leu, Phe and His) were absorbed significantly faster ( P< 0.05 or less) from the low molecular weight lactalbumin hydrolysate (LH1)than from
its equivalent amino acid mixture (Table 3). In
contrast, four amino acid residues (Gly, Ala, Ileu
and Leu) were absorbed sigmflcantly slower from
the higher molecular weight hydrolysate than from
its equivalent free amino acid mixture. Comparison
of absorption rates showed that 14 residues (Asx,
Thr, Ser, Glx, Gly, Ala, Val, Met, Ile, Leu, Tyr,
Phe, His and Lys) were absorbed significantlyfaster
from the low molecular weight lactalbumin hydrolysate, LH1, than from the higher molecular weight
preparation, LH2 ( P < 0.05 or less) (Table 3).
Total nitrogen absorption from the solutions
showed a similar pattern. Whilst there was no difference in percentage absorption between the two
amino acid solutions equivalent to LH1 and LH2,
nitrogen was absorbed from the short-chain
hydrolysate LH1 to a markedly greater extent than
from LH2 (Table 4). There was significantly slower
nitrogen absorption from the higher molecular
weight protein hydrolysate than from its equivalent
amino acid mixture.
The higher nitrogen absorption rate from the
short-chain hydrolysate (compared with the longer
chain hydrolysate) was also mirrored by water, but
not sodium, absorption rates (Table 4). In contrast,
TABLE
3. Percentage absorption of amino acid residues during jejunal perfusion of the
two lactalbumin hydrolysates and their equivalent amino acid mixtures
Results are expressed as percentage absorption of infused load (means k SEM, n = 6).
* P < 0.05, **P< 0.01, hydrolysate compared with free amino acid mixture; t P < 0.05,
tt P < 0.01, hydrolysate compared with hydrolysate.
Low lactalbumin (LH1)
Hydrolysate
Asx
Thr
Ser
Glx
Pro
GlY
Ala
Val
CYS
Met
Ile
Leu
TYr
Phe
His
LYS
Arg
40 f 6**tt
46 f 6*tt
50 -I 5**tt
30 f 3**t
55+2(n=3)
40 f 4*t
57 f 5**tt
65 f 6*tt
27f5
69 f7tt
67 f 7tt
70 f 6*tt
48 f loft
64 f 5**tt
46 f3*tt
44 f5t
28f9
Free amino
acid mixture
1457
21 f 2
26f2
14f2
60+9
29f6
39f4
40f4
30f9
62f8
63f5
57f5
3355
32f2
19f8
39f3
28 f14
Higher lactalbumin (LH2)
Hydrolysate
14f2
18f3
17f3
14fl
26+9(n=3)
15+2*
17 f 2*
21f5
17f6
19f5
17 f 4*
18-+2*
19f2
25f9
25f5
17f4
33 f13
Free amino
acid mixture
20f3
26f4
29f3
14f4
53f16
40f3
46f3
44f7
11f3
51 f 16
64fll
64f4
31 f 15
37f7
21 f 10
39f8
30f9
G. K . Grimble et al.
68
TABLE
4.Absorption of nitrogen, sodium and water f.om two lactalbumin hydrolysales
and their equivalent amino acid mixtures
All values are expressed as means ~ S E M ,n = 6. * P < 0.05,**P< 0.02, ***P< 0.01,
hydrolysate compared with free amino acid mixture; t P < 0.05, t t P < 0.01, hydrolysate
compared with hydrolysate.
~~
Solution
Rate of absorption from test segment
Nitrogen
("4
LH 1
Amino acid mixture
LH2
Amino acid mixture
37.7 f2.89tt
31.3 f3.56
12.1 k 3.12***tt
31.1 f4.24***
sodium uptake from both hydrolysates was greater
than from their equivalent amino acid solution,
whilst water absorption from LH2 was less than
from its equivalent amino acid solution.
Discussion
In earlier intestinal perfusion experiments, differences were found in the handling of four different protein hydrolysates [S, 6, 81. Although it
seemed, at the time, that there could be a number of
explanations for the differences (varying amino acid
composition of the starter proteins, different peptide chain lengths and hydrolysis method used),
direct comparisons of the data could not be made
as the experimental conditions differed in some
respects [8]. More recently in a controlled study, the
native protein and hydrolysis method were both
found to influence absorptive profiles of protein
hydrolysates [9]. In the present study we have
shown that the chain length of the constituent peptides also has an important influence on the absorptive properties of partial enzymic hydrolysates in
man. In this case, 14 out of 17 amino acid residues
measured were absorbed significantly faster from
the low, as compared with the higher, molecular
weight lactalbumin hydrolysate and a similar result
was obtained for total nitrogen absorption. There
was also better absorption of most of the amino
acid residues from the low molecular weight lactalbumin hydrolysate (LH1)
than from its free amino
acid mixture. This would suggest that a significant
quantity of amino acid nitrogen in LH1 was
absorbed in the form of unhydrolysed peptides [ 171.
Recent evidence suggests that uptake of unhydrolysed peptides is restricted to those containing two, three and possibly four amino acid residues
[l-3,181and it would appear likely that the component of LH2 with a chain length greater than four
required hydrolysis at the luminal surface of the
Sodium
Water
(mmol h" 25 an-')
(mlh-' 25 cm-')
18 f lo**
6 f5**
16 f 13***
6 f4***
232 f 16t
196f 16
107 f 14*+
208 f 10*
jejunal mucosa by brush-border peptide hydrolases,
or absorbed pancreatic proteases, before absorption [19, 201. In contrast to the results of the perfusions with LH1, 13 residues were absorbed at
similar rates from LH2 and its free amino acid mixture and the remaining four were actually absorbed
faster from the amino acid mixture. The most likely
reason for these differences is that in the absence of
luminal hydrolysis it is the kinetics of brush border
hydrolysis, rather than the kinetics of free amino
acid or peptide transport, that controls the overall
rate of uptake of amino acid nitrogen from partial
enzymic hydrolysates of whole protein.
These findings have implications where maximal
nitrogen assimilation is required in the rare clinical
situations where both luminal hydrolysis and functional absorptive surface area are substantially
reduced [21].Thus consideration should be given to
administering protein hydrolysates with shorter,
rather than longer, peptide chain lengths, thereby
avoiding the need for brush border peptide hydrolysis. It should be appreciated, however, that higher
loads of amino acid nitrogen were administered
during the present perfusion studies (approx. 1400
pmol/min) than are likely to be given during continuous 24 h enteral feeding (approx. 400 pmoV
min) [22].Further studies are therefore required
before firm recommendations can be made about
what constitutes the most 'physiologically based'
peptide nitrogen source for use in the so-called
'chemically defined' pre-digested diets.
References
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3. Silk, D.B.A. (1981) Peptide transport. Clinical
Science, 60,607-615.
Peptide chain length and jejunal absorption
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