Gastrointestinal
injury and Colostrum
ACKNOWLEDGMENTS
Raymond J. PLAYFORD*, Christopher E. MACDONALD?, Denis P. CALNAN?,
David N. FLOYD?, Theo PODAS?, Wendy JOHNSON?, Anthony C. WICKS?, O.
BASHIR* and Tania MARCHBANK*
*Department of Gastroenterology, Imperial College School of Medicine,
Hammersmith Hospital, Du Cane Road, London W12 0NN, U.K., ?Department
of Gastroenterology, Leicester General Hospital, Gwendolen Road, Leicester
LE5 4PW, U.K., and ?SHS International Ltd., 100 Wavertree Boulevard,
Liverpool L7 9PT, U.K.
Non-steroidal anti-inflammatory drugs (NSAIDs) are effective analgesics
but cause gastrointestinal injury. Present prophylactic measures are
suboptimal and novel therapies are required. Bovine colostrum is a
cheap, readily available source of growth factors, which reduces gastrointestinal
injury in rats and mice.
We therefore
examined whether spray-dried, defatted colostrum could reduce the
rise in gut permeability (a non-invasive marker of intestinal injury)
caused by NSAIDs in volunteers and patients taking NSAIDs for clinical
reasons. Healthy male volunteers (n = 7) participated in a randomized
crossover trial comparing changes in gut permeability (lactulose/rhamnose
ratios) before and after 5 days of 50 mg of indomethacin three times
daily (tds) per oral with colostrum (125 ml, tds) or whey protein
(control) co-administration. A second study examined the effect of
colostral and control solutions (125 ml, tds for 7 days) on gut permeability
in patients (n = 15) taking a substantial, regular dose of an NSAID
for clinical reasons.
For both studies,
there was a 2 week washout period between treatment arms. In volunteers,
indomethacin caused a 3-fold increase in gut permeability in the control
arm (lactulose/rhamnose ratio 0.36㊣0.07 prior to indomethacin and
1.17㊣0.25 on day 5, P < 0.01), whereas no significant increase
in permeability was seen when colostrum was co-administered. In patients
taking long-term NSAID treatment, initial permeability ratios were
low (0.13㊣0.02), despite continuing on the drug, and permeability
was not influenced by co-administration of test solutions. These studies
provide preliminary evidence that bovine colostrum, which is already
currently available as an over-the-counter preparation, may provide
a novel approach to the prevention of NSAID-induced gastrointestinal
damage in humans.
INTRODUCTION
Non-steroidal
anti-inflammatory drugs (NSAIDs) are some of the most commonly prescribed
medicines used worldwide. Although of undoubted efficacy for the treatment
of musculoskeletal injury, chronic administration of NSAIDs results
in both gastric and intestinal damage. This includes peptic ulceration
and injury to the small and large intestine causing increased permeability
with blood and protein loss and stricture formation [1每4].
Current strategies
to reduce gastrointestinal side effects of NSAIDs consist of co-administration
of acid suppressants or prostaglandin analogues. Although they are
beneficial in reducing peptic ulceration, they are less efficacious
in limiting small intestinal damage [3]. In addition, diarrhoea can
be a troublesome side effect from the use of prostaglandin analogues,
and these drugs are also relatively contraindicated in young women
because of their pro-abortive and teratogenic activity [5]. Novel
therapeutic approaches are therefore required.
Colostrum, the
milk produced for the first few days after birth, is a rich natural
source of nutrients, antibodies and growth factors for the suckling
neonate. Some studies suggest it may be of value in eliminating infection
and stimulating growth of the neonatal gastrointestinal tract [6,7].
Its value in the prevention and treatment of adult gastrointestinal
injury is, however, largely unexplored. We have shown recently [8],
using a combination of in vitro and in vivo animal models, that a
commercially available defatted bovine colostral preparation can reduce
NSAID-induced gut injury in rats and mice.
We have now further
examined its potential clinical value for the prevention and treatment
of NSAID-induced enteropathy by measuring changes in gut permeability
in normal volunteers taking clinically relevant doses of the NSAID
indomethacin and also in patients taking long-term NSAIDs for clinical
reasons.
METHODS
Materials and
ethics
Chemicals were
obtained from Sigma unless otherwise stated. Local ethical approval
and patient consent was obtained for all studies.
Preparation of
colostrum and whey protein solutions
The test solutions
were identical to those used for the previously published in vitro
and in vivo studies [8], and were prepared by Viable Bioproducts,
Turku, Finland. The initial colostral and milk whey protein solutions
were treated in an identical fashion and were passed through a microfilter
(0.2 mm pore). The final colostral whey solution ('Bioenervi') is
free of fat (including polar lipids) and lactose, and is reduced in
most of the major milk proteins, including casein and lactalbumin,
with the remaining protein being relatively rich in immunoglobulins
and growth factors.
The total protein
content of the colostral solution was 4.3 mg/ml. The concentrations
of the various growth factors present in the colostral preparation
are incompletely defined, but include: insulin-like growth factor-I
and -II, at approx. 2 mg/l each; transforming growth factor b at 25
米g/l; and epidermal growth factor at 6 米g/l (data supplied by SHS
International Ltd, personal communication). The milk whey (placebo)
solution provided an isoproteinaceous solution (4.3 mg/ml), which
has a similar appearance to the colostral preparation, but is free
of growth factor constituents (data supplied by SHS International
Ltd, personal communication).
Assessment of
permeability
Assessment of
intestinal permeability by quantifying unmediated absorption of at
least two sugars of different sizes provides a sensitive index of
intestinal damage [9]. We used lactulose as the disaccharide probe,
and rhamnose and mannitol as two alternative monosaccharide probes
in a hypo-osmolar formulation. Both rhamnose and mannitol have been
widely used and provide similar information regarding changes in 'paracellular
pathways'. However, as there are minor differences in their processing
in vivo [9], both sugars were included in the test drink.
Following an
overnight fast, subjects emptied their bladders and then drank a standardized
sugar solution containing 5 g of lactulose, 2 g of mannitol and 1
g of rhamnose in a total of 450 ml of water (calculated osmolality
69 osmol/kg of water). Subjects were allowed unlimited intake of fluid
after the first hour of the test to ensure adequate urine output.
The urine was collected and pooled over the next 5 h and total volume
recorded. Aliquots were centrifuged briefly to remove gross debris
and the supernatant frozen at -25 ∼C until later analysis.
Analyses of sugar
content within the urine were based on the method of S?rensen et al.
[10], with minor modifications. The various sugars were separated
using HPLC and quantified using a pulsed amphometric detector. Using
this technique, sugars are oxidized on the gold electrode at the working
potential (E∩0 = 0.05 V), the current produced being a measure of
the amount of sugar present in the sample [11].
The system comprised
of a Hewlett Packard 1100 series HPLC system using a Dionex CarboPac
PA10 anion-exchange analytical column (4.5℅250 mm) with an equivalent
guard column. NaOH (50 mM, low in carbonate; BDH每Merck Ltd) was used
as the mobile phase (1 ml/min, isocratic conditions). Sugars were
detected using a Hewlett Packard 1049A Electrochemical Detector with
a gold working electrode and solid reference electrode. The potentials
were set as follows: E∩0 = 0.05 V, E∩1 = 0.6 V and E∩2 =-0.8 V; and
t1 = 120 ms, t2 = 120 ms, t3 = 400 ms. Data analyses were performed
using the Hewlett Packard Chemstation software.
Typically, the
mannitol peak eluted from the column at approx. 2 min, rhamnose at
4 min and lactulose at 9 min. Analysis of single- and mixed-sugar
standards in the concentration range 0.05 mg/ml to 50 mg/ml showed
good chromatographic separation and proportional changes in the area
under the curve (results not shown).
Initial baseline
urine samples, analysed following the preliminary studies, showed
no sugar peaks in these areas. However, when the same separation protocol
was used for the subsequent full studies, the mannitol peak was sometimes
obscured by an overlap from other urinary constituents (Figure 1).
We therefore used the lactulose/rhamnose ratio as our index of intestinal
injury, a combination that has been recommended for assessing enteropathy
induced by NSAIDs [12].
Study protocols
Normal volunteers
(Study 1)
To determine
the reproducibility of results, a single individual performed permeability
studies for 6 separate days (while not taking any test treatments
or NSAIDs). These samples were assayed to determine intra-patient
variation. In addition, a single sample was measured six times to
determine intra-assay variation.
To examine whether
the colostral or control preparation influenced permeability under
basal conditions, four subjects underwent an initial permeability
assessment and then ingested either the colostral or whey protein
preparation [125 ml, three times daily (tds)] for 5 days with a further
assessment on the final day. No change in permeability was seen (Figure
2).
Seven male volunteers
(26每38 years old) who were not intolerant of milk products, taking
NSAIDs, or suffering from conditions likely to affect intestinal permeability
(e.g. coeliac disease or previous intestinal surgery), were entered
into the study.
Subjects abstained
from alcohol consumption and ingestion of any NSAID, including aspirin,
for 1 week prior to starting the study and throughout the remainder
of the test period. Following an initial baseline permeability assessment,
they received, in random order, the colostral preparation (125 ml,
tds) or control solution for 7 days. For the last 5 days of each study
arm, they also took indomethacin 50 mg, tds. At the end of the test
period intestinal permeability was reassessed. A 2 week 'washout'
period was left between the two stages of the study. Throughout the
study, volunteers and patients were asked about symptoms and compliance.
Formal dyspepsia scoring was not undertaken, however, as the standard
validated questionnaires are not appropriate for short-term studies.
Patient group
(Study 2)
A total of 15
patients (seven male, eight female; mean age 61 years, range 43每75)
were recruited from the community (predominantly from Latham House
Surgery, Melton Mowbray, Leicestershire, U.K.). All had been regularly
taking a stable, substantive dose of a non-selective NSAID without
additional prophylaxis (eg. acid suppressant) for at least 1 year.
This comprised of Voltarol (75 mg or higher) in two patients, Piroxicam
(20 mg or higher) in three patients, Naproxen (500 mg twice daily
or higher) in five patients and Ibuprofen (1.2 g or higher) in five
patients. The underlying conditions necessitating NSAID usage were:
osteoarthritis, in 11 patients; rheumatoid arthritis, in three patients;
and psoriatic arthropathy, in one patient. None of the patients were
taking any other drugs likely to alter intestinal permeability or
had previously suffered any clinical adverse events due to NSAIDs.
Patients did not undergo endoscopy prior to starting the trial.
Patients were
randomized to receive, in a double-blinded, randomized control fashion,
colostrum or control solution (125 ml, tds for 7 days) with a 2 week
washout period between the two study arms. For both arms of the study
(colostrum or control), an initial baseline assessment of intestinal
permeability was performed prior to starting the test solutions. Permeability
was re-assessed after 7 days treatment. Patients were assessed by
telephone interview on day four and at the end of treatment with a
structured questionnaire to document consumption of their NSAID, and
test treatment and identify side effects (if any).
Statistics
Data were analysed
by one- or two-way ANOVA as appropriate, using presence of indomethacin
and test solution as factors. When a significant effect was found
(P < 0.05), individual comparisons were performed using t tests
based on the residual and degrees of freedom obtained from the ANOVA,
a method equivalent to repeated-measures analyses, which takes account
of the fact that the data are paired. Comparison of baseline values
of Study 1 versus initial values of patients in Study 2 were performed
using a two-tailed unpaired t test.
RESULTS
Normal volunteers
(Study 1)
For multiple
measurements of a single sample, the coefficient of variation was
5.9%. Measurement of the six serial samples from the same individual
gave a coefficient of variation of 7.7%. Administration of colostrum
or control solution to the individuals not taking NSAIDs had no effect
on their permeability results (Figure 2). Therefore although there
was a relatively broad normal range (0.17每0.81, see Figure 2 and baseline
values of Figure 3), in keeping with other published works (for example
[3]), intra-volunteer variation was small. It was for this reason
that each subject acted as their own control.
All seven subjects
completed the study without protocol violations. One developed mild
nondescript upper-abdominal discomfort while taking the indomethacin
(control) arm but completed the course. No side effects due to ingestion
of the colostral preparation were reported.
Baseline permeability
values were similar at the beginning of each study arm (lactulose/rhamnose
ratio 0.36㊣0.07 versus 0.42㊣0.10, means㊣S.E.M., P > 0.05; Figure
3). Permeability increased approx. 3-fold in response to indomethacin
in the control arm (P < 0.01 versus baseline value), but showed
no significant rise when colostrum was co-administered (Figure 3).
The order in which control and colostrum were administered did not
appear to influence results (although numbers are too small to perform
detailed statistical analysis).
Patient group
(Study 2)
One subject developed
a non-specific viral-type illness during the washout period and was
therefore withdrawn from the study. Of the remaining patients, one
developed mild oesophageal reflux-type symptoms during their placebo-treatment
week. A second patient reported improvement in long-standing vague
lower-abdominal pain while taking colostrum.
Baseline permeability
assessments (assessed while patients continued to take their regular
NSAID therapy) gave results which were actually lower than those seen
in the volunteers of Study 1 prior to them starting indomethacin (lactulose/rhamnose
ratio 0.13㊣0.02, means㊣S.E.M., in patients compared with 0.36㊣0.07
volunteers, P < 0.05). There was however, some overlap between
individuals in the two studies. Co-administration of the colostral
or control solution to patients taking NSAIDs had no significant effect
on their gut permeability (Figure 4).
DISCUSSION
We have used
changes in gut permeability, a well validated indirect method of investigating
small intestinal injury, to examine the potential clinical value of
a commercial defatted colostral preparation in reducing NSAID-induced
enteropathy. The colostral preparation, but not a similarly prepared
whey protein solution, significantly reduced the increase in permeability
caused by short-term (5 day) exposure to indomethacin in normal subjects.
Patients taking long-term NSAIDs for clinical reasons had initial
permeability values that were low, being similar or lower than those
seen in normal subjects not given NSAIDs, and were not influenced
by co-administration of the colostral preparation.
Several methods
are available to determine the degree of small intestinal injury induced
by NSAIDs, all of which have their drawbacks: enteroscopy is an invasive
procedure; 111In-labelled white cells require radioactive exposure;
and measurement of the neutrophil marker, calprotectin, in the stool
is still at a relatively early stage of development [13]. Measurement
of gut permeability is a safe and simple investigation to perform,
but is an indirect method of assessing small intestinal injury. Assessment
of excretion of two molecules of different sizes, such as a monosaccharide
and a disaccharide, by HPLC with pulsed amphometric detection, provides
high sensitivity and allows correction for potential confounding factors,
such as changes in the rate of gastric emptying and small intestinal
transit.
Measurement of
intestinal permeability has been used previously to assess the degree
of small intestinal damage in patients with coeliac disease [14] and
Crohn's disease [15], as well as injury caused by NSAIDs. Several
studies have shown that short term (1每7 days) administration of clinically
relevant doses of NSAIDs, such as indomethacin, naproxen and ibuprofen,
increase gut permeability by approx. 3-fold (e.g. [16]). Our results
from the control arm, using normal volunteers, are therefore in keeping
with published works.
NSAIDs are one
of the most widely prescribed group of drugs used worldwide. Point-prevalence
studies, however, suggest that 10每30% of unselected patients taking
NSAID therapy have peptic ulceration [17], which can often be asymptomatic
[18]. In addition, up to 70% of patients taking NSAIDs have some degree
of enteropathy associated with low-grade blood and protein loss [13,19每21],
although it is only of clinical significance in a much smaller percentage
of patients. Specific cyclooxygenase-2 inhibitors have reduced gastric
toxicity but are expensive and cannot replace the use of aspirin as
an anti-platelet agent, the use of which continues to be a major contributor
to the development of significant gastrointestinal bleeding [22,23].
In the present
study, the baseline permeability values of patients taking long-term
NSAIDs were low, being similar or lower than those seen in control
subjects not given an NSAID. This result is in keeping with the report
of Struthers et al. [24], but is at variance with the report of Sigthorsson
et al. [25] who found an approx. 2-fold increase in permeability in
patients taking similar doses of NSAIDs to those in the present study.
The reasons behind these different results are unclear, although variations
exist in the details of the probes used and the osmolality of the
test solutions. We specifically ensured that our test solution was
hypo-osmolar, as this has been reported to maximize alterations in
permeability changes induced by NSAIDs [25]. As all of the volunteers
in the short-term study showed a marked rise in permeability, it is
possible that the small intestine of our patients taking NSAIDs chronically
underwent adaptation. Adaptation is a well recognized phenomenon,
with regards to the disappearance of gastric erosions, in patients
who continue to take NSAIDs [26]. All of our patients were selected
on the basis of having taken NSAIDs long-term, without the requirement
for additional prophylaxis. It is therefore possible that those patients
who suffer serious side effects (such as chronic blood loss) fail
to adapt appropriately. Alternatively, because of the selection criteria,
our patients may represent a group who are not susceptible to the
initial effects of the NSAID which were seen in the volunteers. This
idea is less likely, however, as although the number of volunteers
were small, all responded in a similar manner when acutely exposed
to indomethacin. Additional differences existed between the patients
and volunteers; the mean age of patients was greater than that of
volunteers and consisted of both males and females. This last point
was probably not of major relevance as sub-analyses of the males alone
showed similar results. To address this area further, additional larger
studies should probably examine gut permeability in patients prior
to starting NSAID therapy, with serial measurements of side effects
(dyspepsia scores etc.) and permeability following randomization to
control, or colostrum solution, at the same time as NSAID therapy
is started.
Colostrum is
the milk produced by the mother for the first few days after birth
and is much richer in growth factors and antibodies than ordinary
milk [6,7,27]. Bovine colostral preparations are currently available
in the U.S.A. and throughout Europe as 'over-the-counter' health food
supplements. They do, however, contain large amounts of potent growth
factors which are biologically active [8]. Products such as these
are also termed 'functional foods' or 'nutraceuticals' based on the
realization that the distinction between food and drugs is becoming
blurred.
NSAIDs such as
indomethacin cause damage to the gastrointestinal tract by several
mechanisms, including reduction of mucosal prostaglandin levels, reduction
of mucosal blood flow, stimulating neutrophil activation and, possibly,
also stimulating apoptosis [28]. It is likely that many of these mechanisms
will be influenced by the numerous growth factors present in the colostrum
preparation. There is now increasing evidence that administration
of multiple peptides can result in additive or synergistic activity
[29]. Orally administered colostrum-derived preparations, therefore,
appear to be an attractive therapeutic option as they contain multiple
growth factors in a formulation that provides inherent protection
against proteolytic digestion. Further, long-term clinical studies
appear warranted to examine its value in the prevention/treatment
of NSAID-induced gut injury, and also possibly for other ulcerative
conditions of the bowel, such as necrotizing enterocolitis and inflammatory
bowel disease, where therapies are sub-optimal and novel approaches
are required.
ACKNOWLEDGMENTS
We thank the Wellcome Trust and the Medical Research Council for funding.
Declaration of
interest
The use of bovine
colostrum for the prevention of NSAID-induced gut injury has been
patented by SHS International Ltd (No. 9619634.0), who partially funded
this work.
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Received 10 January 2001; accepted 9 March 2001
The Biochemical Society and the Medical Research Society 2001
Articles
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