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Surgical Research of the Department of General Surgery and Thoracic Surgery of the University of Kiel, Michaelisstrasse 5, D-24105 Kiel, Germany,
*
Rheumatism Clinic, Prague, Na Slupi 4, 12850 Prague 2, Czech Republic, and
DGF STOESS AG, 69402 Eberbach, Germany
1To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • gelatin hydrolysate • peptide absorption • organ distribution • cartilage • mice (C57/BL)
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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From a nutritional point of view, gelatin is considered to be a protein
of lower quality since nearly all essential amino acids are either
underrepresented or, in the case of cysteine, completely absent
(Eastoe and Leach 1977
).
Medically, gelatin has been in use for therapeutic purposes for a long
time. In several studies, a positive effect with regard to the growth
of hair and nails was demonstrated subsequent to oral administration of
gelatin hydrolysate (Brodie 1984
, Gehring 1992
, Morganti and Randazzo 1984
). In recent
years, improvement of degenerative joint disease after oral
administration of gelatin hydrolysate received increasing attention. In
addition to animal experiments, clinical investigations also
demonstrated a positive therapeutic effect after orally administered
gelatin hydrolysate, e.g., in the treatment of osteoarthritis
(Adam 1991
, Seeligmüller and Happel 1993
). The question as to gelatin absorption from the gut and
its therapeutic mechanism remain essentially unsolved. Apart from a
metabolic stimulation leading to an increase of collagen synthesis
(Lippiello et al. 1977
, Nagler-Anderson et al. 1986
), a modulation of immunological processes was also
discussed (Adam 1991
, Trentham et al. 1993
). Before speculating about the mechanism of the
therapeutic effectiveness of gelatin hydrolysate, the question must be
clarified as to whether gelatin hydrolysate can be absorbed from the
intestine and furthermore in what form and quantity. In this
investigation the time course of gelatin hydrolysate absorption was
studied as well as its subsequent distribution in various tissues and
organs. This study also included an investigation into the differential
enrichment by certain tissues of gelatin hydrolysate or separately
administered proline.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The 14C labeled gelatin hydrolysate was produced by intraperitoneal application of 14C-proline to Wistar rats (Animal Service, Charles University, Prague, Czech Republic). The skins of these animals were collected and hydrolyzed (50 min at 50°C) by enzymes (DGF Stoess, Eberbach, Germany) to obtain 14C labeled gelatin hydrolysate with a mean MW of 6.1–6.8 kD. The specific radioactivity was 57.4 kBq/g. Uniformly labeled 14C-proline was obtained from Hartmann Analytic (Braunschweig, Germany), with a specific activity of 8.3 GBq/mmol. Before administration, 14C-proline was diluted with 9 g/L NaCl to achieve preparations with the desired radioactivity. Gelatin hydrolysate (batch no.: 830814) was obtained from DGF Stoess (Eberbach, Germany).
Animal model.
The experiments were performed on male mice (C57/BL; Harlan Winkelmann, Paderborn, Germany) with a body weight of 21–25 g. The animals were housed under standardized conditions with free access to nonpurified diet (Altromin standard diet 1310, containing 22% crude protein, 5% crude fat, 4% crude fiber average content; Altomin GmbH, Lage, Germany) and water. At the beginning of the experiment, the animals were randomly assigned to either the control or the test group. The test substances were administered via a gastric feeding tube. Mice of the gelatin group received 10 mg of 14C labeled gelatin hydrolysate/g body weight (580 Bq/g body wt). In the control group 14C labeled proline (580 Bq/g body wt) along with unlabeled gelatin hydrolysate (10 mg/g body wt) was administered. Thus all animals received standard doses of radioactivity of 580 Bq/g and 10 mg of gelatin hydrolysate/g body wt, respectively. Both before and after administration of the above substances, the animals had free access to nonpurified diet and water. The mice from each group were sacrificed 3, 6, 12, 24, 48, 96 and 192 h after oral administration, and samples of blood and various types of tissues and organs were collected. A volume of 0.6 mL of blood (35 IU heparin/mL) was taken from the portal vein. From each animal plasma, cartilage, skin, liver, kidney, skeletal muscle and spleen were removed, and radioactivity of the samples was measured in aliquots. Furthermore, the remaining 14C-activity in the whole gastrointestinal tract was analyzed after the end of absorption time to determine the intestinal uptake of the test substances.
This study was approved by the Animal Research Committee at Kiel University. Care and handling of the animals were in accordance with the National Institutes of Health guidelines.
Sample preparation and measurement of radioactivity.
The radioactivity of plasma samples was determined following the addition of liquid scintillator Aquasafe 300 Plus (Zinsser Analytic, Frankfurt/Main, Germany), using polystyrene scintillation vials (Zinsser). Tissues were minced with scissors, and weighed aliquots of each tissue were digested in Biolute S (Zinsser Analytic) for 12 h at 50°C. Samples were bleached with hydrogen peroxide (30%) and radioactivity determined following the addition of Aquasafe 300 Plus.
For the measurement of radioactivity, a liquid scintillation counter (Packard Tricarb 2000 CA; Camberra-Packard, Dreieich, Germany) with external standardization was used. All analyses were conducted in triplicate taking the respective background values into account. Counting was performed for 15 min or up to a statistical confidence level of 2 sigma error equal or less than 5%. The counting efficiency was 88–93% for all samples.
Gut-sac technique.
Qualitative investigations of the absorption of the gelatin hydrolysate
were carried out using the "gut-sac" method (Schilling and Mitra 1990
, Wilson and Wisemann 1954
). C57/BL
mice were anesthetized by an i.p. injection of 90 mg/kg body wt
ketamine hydrochloride (Ketanest; Parke-Davis Pharmaceutical
Research, Morris Plains, NJ). After making a midline incision in the
abdomen, 4–6-cm pieces of small intestine were removed beginning 5-cm
distal to the stomach. The pieces of intestine were rinsed, closed on
one end and filled with 0.8 mL of gelatin hydrolysate solution (500
g/L). The end of the segment was ligated with silk, and then the gut
sac was immediately immersed in the test tube containing 5 mL of 37°C
9 g/L NaCl solution (absorption medium) and continuously bubbled with
95% O2/5% CO2. After 30 min, an aliquot of
the absorption medium was removed for subsequent analysis of the
protein profile by SDS gel electrophoresis and HPLC. In control
experiments the intestinal segments were filled with 9 g/L of NaCl
solution.
Electrophoresis.
A discontinuous SDS-PAGE was performed as described by
Schägger and Jagow (1987)
with 16% acrylamide in
the separating gel using a Mini-Protean II Cell (Bio-Rad
Laboratories, München, Germany). Gels were stained by the method
of Heukeshoven and Dernick (1985)
. The MW markers of
2.5–17 kD were obtained from Sigma-Aldrich (Deisenhofen, Germany).
HPLC.
GPC–HPLC measurements were performed using a Pharmacia LKB 2150 system equipped with a UV-detector operating at 214 nm and a column TSK 2000 SW (XL) (Haas, Heidelberg, Germany) with 200 mmol/L of sodium phosphate buffer, pH 5.3, as solvent.
Statistical analysis.
All results are expressed as mean values ± SEM. The significance of differences in values was assessed by the Mann-Whitney U-test. Differences with P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Subsequent to oral administration of 14C labeled gelatin
hydrolysate, a rapid increase of radioactivity was observed in plasma,
reaching a maximal concentration 6 h after the beginning of the
observation period (Fig. 1
). This peak was followed by a marked decrease of radioactivity. After
beginning of the experiment (24 h) more than 85% of radioactivity in
plasma disappeared. Later (3 d) the radioactivity declined to values
near the limits of detectability in the gelatin group.
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) and, in contrast to plasma, 14C-activity remained
relatively high (467 ± 107 dpm/100 mg) up to 96 h. At the
end of the observation period (192 h), measured radioactivity declined
to ~58% of the peak value. In plasma as well as in tissues and
organs under investigation (Table 1
), radioactivity revealed no significant differences between the values
obtained following administration of 14C labeled gelatin
hydrolysate and the control group animals, which had received
14C-proline together with unlabeled gelatin hydrolysate.
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). As early as 12 h after the beginning of the observation period,
radioactivity in cartilage was significantly (P < 0.05) higher in mice which had received 14C labeled gelatin
hydrolysate than in control animals. In the experimental group, a
pronounced increase in radioactivity could be observed reaching a peak
value (246 ± 34 dpm/100 mg) at 48 h. At 96 h,
~2.6-fold more radioactivity was detected in cartilage from the
gelatin group compared with the control group. At the end of the
observation period, only low radioactivity could be determined in
cartilage of both groups. Investigations of the radioactivity remaining
in gastrointestinal tract revealed a rapid and pronounced depletion of
14C-activity in the intestine, which correlated well with
the initial increase of radioactivity in plasma as well as in all
tissues. After 6 h of absorption time, a residual activity of
<10% of the initial activity was determined in the gastrointestinal
tract in both groups. Subsequent to administration (12 h) of the test
substances, more than 95% of the administered radioactivity had been
absorbed. The amount of the remaining radioactivity did not differ
significantly in both groups.
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To gain more information about the qualitative absorption of gelatin hydrolysate in the intestinal tract, "gut-sac" experiments were performed.
Subsequent to absorption of gelatin hydrolysate, proteins in a range
from 2.5 kD to <15 kD MW were detected in the absorption medium
(n = 4) by means of SDS electrophoresis (Fig. 4
). The determined protein profile corresponded to the protein profile of
gelatin hydrolysate (<1–25 kD) with a mean M.W. of 3.5 kD. A marked
difference could be observed compared to the control experiments
(absorption of 9 g/L of NaCl solution) despite the insufficient
separation of the proteins. Subsequent to absorption of NaCl solution,
almost no proteins of M.W. <25 kD were detected in the absorption
medium.
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). In the control group without gelatin absorption, no proteins of MW <10
kD could be determined.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and
Laser-Reutersward et al. (1985)
concerning the
digestibility of collagen. In addition to the fact that gelatin
hydrolysate is absorbed from the intestine, it is of considerable
importance to analyze the quality of gelatin uptake. It is commonly
assumed that peptides are hydrolyzed in the gastrointestinal tract
prior to absorption, so that free amino acids enter the circulation
predominantly (Boullin et al. 1973
, Mathews and Laster 1965
). In contrast, however, there is considerable
evidence that peptides or even macromolecules can also be absorbed
intact (Seifert and Sass 1990
, Urao et al. 1997
, Warshaw et al. 1974
). Earlier
investigations suggested that both peptides (Gardener 1982
, Gardener 1983
, Matthews 1975
, Webb 1986
) and macromolecular proteins
(Hemmings and Williams 1978
, Seifert et al. 1979
) are absorbed from the gut and retain some biological
function (Atisook and Madara 1991
, Castell et al. 1997
, Gardener et al. 1991
, Hara et al. 1984
). More recently, investigations of the mechanism of
peptide (Daniel 1997
) and macromolecule (Terpend et al. 1998
) absorption and the influence on immune system
response (Castro and Arntzen 1993
, Kagnoff 1993
, Pusztai 1993
) suggested a therapeutic
effect for orally applied peptides and proteins (Trentham et al. 1993
, Weiner 1997
). In the "gut-sac" experiments, we identified peptides within a MW range from 1 kD to ~10 kD on the serosal side of the intestine after the application of gelatin hydrolysate, indicating that gelatin is also absorbed in the high molecular form to some extent. Peptides measured in the absorption medium with a MW of more than 10 kD seem to be of intestinal origin since they could be detected in all gut-sac experiments and controls, respectively. These results could be confirmed by SDS-electrophoresis as well as by HPLC. Although the interpretation of the gut-sac experiments is limited, due to the method, it is evident that in addition to degradation of gelatin and subsequent amino acid uptake, a significant amount of gelatin hydrolysate is absorbed in the high molecular form.
With regard to the distribution of radioactivity in the various tissues
and organs, our current results demonstrate that almost no differences
could be observed after the administration of 14C labeled
gelatin hydrolysate and 14C labeled proline, respectively.
In cartilage, however, a significantly (P < 0.05)
higher degree of radioactivity could be determined for at least 96 h subsequent to administration of labeled gelatin than was the case
after the application of 14C labeled proline. The
distribution of proline in cartilage after oral administration is
consistent with observations of Tonna et al. (1971)
.
Whereas for proline only a slight and transient uptake of
14C-activity could be observed in cartilage, accumulation
of radioactivity was more pronounced after the administration of
gelatin hydrolysate persisting over a longer period than after the
application of proline. The possible reason for this phenomenon might
be due to the fact that, subsequent to intestinal absorption of gelatin
hydrolysate, a heterogeneous mixture of peptides and, to some extent,
polypeptides enters the circulation, and that these peptides are
preferably accumulated in cartilage. Another explanation of the
accumulated values in cartilage could be a reduced metabolic rate of
peptides as well as polypeptides compared to amino acids per se. If
this is the case, similar differences in radioactivity should be
expected in liver, the main organ of metabolism. Since the determined
14C-activity in the liver of animals treated with gelatin
hydrolysate is not different from the radioactivity in animals treated
with proline, it must be concluded that differences in metabolism could
not be responsible for the accumulation in cartilage.
Although the beneficial effect of orally administered gelatin
hydrolysate in the improvement of degenerative joint diseases could be
demonstrated in several investigations, the therapeutic mechanism
remains unsolved. One possible mechanism might involve the modulation
of immunological processes. For the treatment of rheumatoid arthritis,
it was shown in clinical trials that the oral administration of
cartilage-derived type II collagen can improve the symptoms
(Barnett et al. 1998
, Trentham et al. 1993
). This positive effect was explained by an orally induced
tolerance in which mainly T-helper cells are involved
(Kalden and Sieper 1998
). In order to generate this
tolerance in immunocompetent cells, high-M.W. peptides would have to be
absorbed from the intestine. In addition to the immunological
modulation, a metabolic stimulation was also discussed in connection
with the therapeutic efficacy of orally administered gelatin
hydrolysate. The pronounced accumulation of radioactivity in cartilage
subsequent to administration of gelatin observed in the present study
might possibly be caused by a selective modification of cell
metabolism. In summary, this study indicates that gelatin hydrolysate
is absorbed from the intestine and preferentially accumulated in
cartilage. These results suggest that the unique amino acid and peptide
profile of gelatin may be responsible for the previous observations of
therapeutic efficacy of orally administered gelatin in degenerative
conditions of the musculo-skeletal system (Adam 1991
, Seeligmuller and Happel 1993
). Additional
research is needed to identify the mechanisms responsible for the
preferential accumulation of gelatin hydrolysate in cartilage tissue.
Manuscript received January 26, 1999. Initial review completed March 27, 1999. Revision accepted July 14, 1999.
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