Prof. R. J. Playford, Centre for Gastroenterology, Institute of Cell and Molecular Science, Barts & The London School of Medicine, Queen Mary University of London, Turner Street, London E1 2AD, UK. E-mail: firstname.lastname@example.org
Background A partially hydrolysed and dried product of pacific whiting fish is marketed as a health food supplement supporting ‘intestinal health’.
Aim To examine whether the partially hydrolysed and dried product of pacific whiting fish influenced the small intestinal damaging side effects of the nonsteroidal anti-inflammatory drug, indomethacin.
Methods Eight human volunteers completed a double-blind, placebo-controlled, crossover protocol of clinically relevant dose of indomethacin (50 mg t.d.s. p.o. for 5 days) with 7 days of fish hydrolysate or placebo starting 2 days prior to indomethacin. Changes in gut permeability were assessed using 5 h urinary lactulose:rhamnose (L/R) ratios.
Results Fish hydrolysate given alone did not affect permeability. In the main study (n = 8), baseline values were similar for both arms (0.28 ± 0.05 and 0.35 ± 0.07). Administration of indomethacin (+placebo) caused a fivefold rise in L/R ratios (increasing to 1.54 ± 0.35), whereas L/R ratios in the same subjects ingesting indomethacin + fish hydrolysate was only 0.59 ± 0.14 (P < 0.01 vs. indomethacin alone). Dyspeptic symptoms occurred in four of eight subjects taking indomethacin alone, but zero of eight when hydrolysate was co-administered.
Conclusion Natural bioactive products (nutriceuticals), such as fish hydrolysates, may provide a novel approach to the prevention and treatment of NSAID-induced and other gastrointestinal injurious conditions.
There is currently a resurgence of interest in the use of natural bioactive products (‘nutriceuticals’) among the general public, with many healthy subjects and patients taking them for prevention and treatment of multiple conditions including gastrointestinal disorders.1 Unfortunately, current evidence of the scientific validity of many of these traditional and commercial compounds is severely limited.
One product of interest, that is already commercially available, is a fermented fish product derived from the controlled proteolytic yeast fermentation of pacific whiting (Merluccius productus). Fermentation is a commonly used process in the standard food industry as well as in the bioactive food (nutriceutical) field. Fermentation of foodstuffs has many effects, including partial degradation of protein constituents which, in addition to potentially aiding absorption from the gut, may influence its biological activity.
Fish hydrolysate is claimed to be beneficial for a variety of gut conditions and we have shown it to be capable of stimulating proliferation and migration (restitution) of HT29 cells in vitro.2 However, data examining its ability to influence gut integrity in human studies are severely limited. We chose to examine the potential activity of this fish hydrolysate against the damaging effects of the nonsteroidal anti-inflammatory drug (NSAID) indomethacin, as NSAIDs are some of the most widely prescribed group of drugs worldwide. Despite the undoubted efficacy of NSAIDs, side effects, including peptic ulceration and small intestinal injury are common and novel therapies are still required.3
We, therefore, performed a series of studies to analyse whether ingesting this fish hydrolysate product could influence the damaging effect of indomethacin on small intestinal integrity, as assessed by following changes in permeability (leakiness) of the small intestine in a human clinical trial.
Materials and methods
All chemicals were purchased from Sigma (Poole, Dorset, UK) unless otherwise stated.
Human studies were approved by appropriate regulatory authorities. Clinical trial REC reference number 05/Q0406/97.
Hydrolysed fish protein concentrate
The dried fish protein hydrolysate preparation studied, Seacure, was donated by Proper Nutrition, Inc., Reading, PA, USA (for details of preparation and constituents, see Ref. 2). The fish protein hydrolysate contains 75–80% protein constituents (60% peptides and 40% amino acids) and the major amino acid constituents are glutamine (approximately 14–16% of total), asparagine (10%) and lysine (10%) and 6–10% fish oils.
A placebo was produced that was identical in colour and smell using rice flour with 1% sea cucumber.
Background to method. Assessment of intestinal permeability by quantifying unmediated absorption of at least two sugars of different sizes provides a sensitive index of intestinal damage.4 To maintain consistency with our previous studies (including osmolality), we administered a lactulose, rhamnose, mannitol mixture but again only present lactulose/rhamnose ratios for reasons previously described.5 This is considered appropriate, as this specific combination has been recommended for assessing enteropathy induced by NSAIDs.6
Protocol. Following an overnight fast, subjects emptied their bladders and then drank a standardized sugar solution containing lactulose 5 g, mannitol 2 g and rhamnose 1 g in a total of 450 mL water (calculated osmolality 69 mOsm). 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 was frozen at −20 °C until later analysis.
Analyses. Analyses of sugar content within the urine were based on the methods and equipment used by our group previously.5 The various sugars were separated using high-pressure liquid chromatography and quantified using a pulsed amphometric detector. Using this technique, sugars are oxidized on the gold electrode at the working potential (P = 0.05 V), the current produced being a measure of the amount of sugar present in the sample.7
Preliminary studies. (i) To determine the reproducibility of results, a single individual performed permeability studies for 4 separate days (while not taking any test treatment or NSAIDs). These samples were assayed to determine intra-patient variation and a coefficient of variation of about 8% was obtained. In addition, a single sample was measured six times to determine intra-assay variation and a coefficient of variation of 5% was obtained. These coefficients of variation were similar to those reported by us previously.5
(ii) To examine whether fish hydrolysate influenced permeability under basal conditions, four subjects underwent an initial permeability assessment and were then ingested fish hydrolysate for 7 days with a further assessment on the final day.
Main study. Ten volunteers (25–40 year, 5M, 5F) who were not taking NSAIDs or suffering from conditions likely to affect intestinal permeability e.g. coeliac disease or previous intestinal surgery, were enrolled 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. Subjects were asked to record and report if they had any upper abdominal discomfort (‘dyspepsia’) prior to starting the protocol or during the two arms of the study.
Each subject underwent a total of six permeability assessments (Figure 1). Each arm of the study comprised three collections on days −2, 0 and 7. For both arms, samples collected on days −2 and 0 were ‘baseline’ analyses when the volunteer was not taking any test substance. After urine collection on day 0, volunteers took test substance comprising fish hydrolysate (1 g) or placebo capsule three times daily for a week. Fish hydrolysate and placebo capsules were indistinguishable in terms of appearance and taste.
After the week’s course of test substance (day 7), the third urine collection for that arm of the study was completed. Following a 2-week washout period, each participant repeated the protocol with the other test capsule. The active and placebo capsule arms of the study were administered in random order. In addition, in both arms of the study, for the last 5 days of the study (days 2–7) participants also received an NSAID (indomethacin 50 mg t.d.s., see Figure 1).
All values are expressed as the mean ± S.E.M. Two-way anova was used with presence of NSAID and presence of fish hydrolysate as factors. Where a significant effect was seen (P < 0.05), individual comparisons were performed using t-tests based on the group means, residual and degrees of freedom obtained from the anova, a method equivalent to repeated-measures analyses.
In the four subjects who took fish hydrolysate for 7 days without taking an NSAID, there was no change in intestinal permeability (lactulose:rhamnose ratio was 0.53 ± 0.13 before treatment vs. 0.54 ± 0.13 after treatment; Figure 2).
Withdrawal. Two female volunteers (who started on the indomethacin plus placebo phase) were unable to tolerate the indomethacin because of onset of abdominal discomfort and withdrew from the study within 2 days of starting. They have, therefore, not been included in the analyses. The remaining eight subjects completed the study.
Dyspepsia. At the beginning of the study, seven of eight subjects who completed the study had no dyspeptic (abdominal discomfort) symptoms and one subject had longstanding mild dyspepsia. There was no worsening or onset of symptoms in any of the eight subjects when they took indomethacin plus fish hydrolysate. In contrast, four of eight complained of onset or worsening of their dyspeptic symptoms when taking the indomethacin with placebo.
Permeability. Baseline permeability values were similar at the beginning of each study arm and at the first and second (baseline) assessments within a study arm (Figure 3). Permeability increased about fivefold in response to indomethacin during the placebo arm (rising from 0.28 ± 0.05, initial baseline value, to 1.54 ± 0.35 at day 7, P < 0.01). In contrast, when the subjects were also taking the fish hydrolysate, the rise in permeability caused by indomethacin was virtually completely abrogated in seven of eight of the subjects and was markedly truncated in the eighth individual (rising from 0.35 ± 0.07, initial baseline value, to 0.59 ± 0.14 at day 7). Statistical analyses, using presence of fish hydrolysate and time as factors showed significant effects of time (i.e. indomethacin administration, F2,42 = 13.69, P < 0.0001), presence of fish hydrolysate (F1,42 = 4.34, P = 0.0433) and an interaction between the two (F2,42 = 6.27, P = 0.004). This showed that the rise in intestinal permeability caused by indomethacin was truncated by the presence of fish hydrolysate. Subsequent t-test comparisons based on mean square error of residual from anova showed that permeability values at day 7 of both arms of study were significantly different (P < 0.01). The order in which placebo and fish hydrolysate was administered did not influence results (although numbers are too small to perform a detailed statistical analysis).
We have shown, for the first time, a commercially available fish hydrolysate preparation reduces the degree of small intestinal damage caused by the NSAID indomethacin in a pilot human clinical trial.
Several methods are available to determine the degree of small intestinal injury induced by NSAIDs in humans, 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.8 Measurement of gut permeability is a safe, sensitive and simple investigation to perform but, as for all the other methods of assessment that do not involve direct visualization, it is not readily equated to a score that uses visualization, such as the number of erosions.9 Measurement of intestinal permeability has been used to assess the degree of small intestinal damage in patients with coeliac disease and Crohn’s disease10 and NSAID11 usage. The fivefold rise in permeability found in the control arm of our main study is in keeping with our,5, 12 and others,11 published works.
Nonsteroidal anti-inflammatory drugs are used worldwide, but side effects such as dyspepsia, peptic ulceration and enteropathy are common and only partially relieved by acid suppressants. Therefore, there is a need for novel therapies. Indomethacin causes damage to the gastrointestinal tract by several mechanisms including reduction in mucosal prostaglandin levels, reduction in mucosal blood flow, stimulating neutrophil activation and stimulating apoptosis.13 It is likely that many of these mechanisms will be influenced by the numerous factors present in the fish hydrolysate. We previously demonstrated pro-proliferative and pro-migratory (restitutive) activity of this fish hydrolysate product in vitro,2 and the current studies expanded these in vitro results to show this product’s efficacy in a clinically relevant model.
The molecules responsible for the protective effects of the hydrolysate against indomethacin-induced injury seen in this study are incompletely defined. It is unlikely that this effect was caused by the hydolysate influencing indomethacin adsorption as we showed previously that the hydrolysate can reduce (systemically administered) indomethacin-induced gastric injury in rats.2 Our previous studies suggested that glutamine within the fish hydrolysate played an important part, accounting for about 34% of the pro-proliferative activity seen using an in vitro model and may also have contributed to antioxidant activity via stimulation of glutathione production. Many of the amino acid constituents of the fish protein hydrolysate remain in the intact protein or partially cleaved form and it is also possible that these intact proteins have direct bioactivity as shown in partially digested bovine milk.14 The fish hydrolysate contains about 6–10% fish oils.2 In addition to increasing migration15 and affecting prostaglandin production, these oils may contribute to anti-apoptotic effects.16 Further work is required, however, to determine in detail how these effects are mediated.
Hundreds, if not thousands, of products are currently marketed as ‘health food supplements.17 72% of the American population uses one or more health supplement products regularly and 57% considered that these therapies reduced their need for drugs and other medical therapies. This results in an annual turnover of £9 billion in the US and £1.2 billion in the UK, with an 8% annual growth.17 The sources of these ‘natural’ products are diverse and include bacteria, plant, animal and marine origins.1, 18 Unfortunately, current evidence of the scientific validity of most of these traditional and commercial compounds is severely limited and the level of evidence used in support of their claims often falls well below that acceptable in the medical and scientific community.
The fish hydrolysate studied by us is already marketed in the US as an ‘over the counter’ health food supplement and, as with many of these products, its major marketing strategy is via patient testimonials. There have, however, been some limited scientific studies. For example, Englender et al.19 reported that use of the fish protein hydrolysate, at a dosage of 3 g/day for 60 days, reduced the symptoms of occasional diarrhoea and constipation and alleviated bloating.
The positive effect in this study is of particular interest as the doses of NSAID and fish hydrolysate used are at standard clinical dose and manufacturers recommended dose, respectively. These results emphasize that the division between ‘food products’ and ‘drugs’, when considered in terms of biological activity (and concerns about potential safety aspects), is far from clear and products such as this should be considered as a ‘nutriceuticals’ or functional foods. Although not directly relevant for this fish hydrolysate product, the distinction between ‘natural’ and ‘artificial’ is also blurred, if a single compound is isolated and clinically used, even though it was from a ‘natural’ source initially.
Further study of products based on fish hydrolysate for the prevention and treatment of other injurious conditions of the bowel, such as inflammatory bowel disease, necrotizing enterocolitis, chemotherapy-induced mucositis, etc., where therapy is suboptimal and novel approaches are required and appear justified.
Declaration of personal interests: None. Declaration of funding interests: This work was partially funded by Proper Nutrition, Inc., who are the manufactures of the Seacure product used. They were not involved with the data analyses. This work was partially funded by the Wexham Park Gastrointestinal trust grant number 2004/6772.