Creatinine, a product constantly produced in muscles from the breakdown of creatine, is accumulated in the blood when renal excretory functions are impaired. This build up is indicative of late stage chronic renal failure and must be removed. Currently, dialysis offers an excellent treatment modality for chronic renal failure and end stage renal disease (ESRD) patients to lower creatinine and other unwanted metabolites. Dialysis, however, has substantial limitations. For example, it is associated with complications such as cardiomyopathy-related deaths (1) and peritonitis (2) particularly in children where the catheter has been in place for a long time (3). Although rare, surgical complications such as catheter site hemorrhages and intestinal perforation may also occur (4). As dialysis is a life-long treatment, it has been shown to also have negative psychological effects, inducing depression and anxiety in patients (5).
Despite constant advances in dialysis technology such as high-efficiency and high-flux dialysis, the rate of mortality and morbidity of kidney disease remains high (6). This has prompted researchers to shift away from improving dialysis methods to try other methods to treat renal failure, such as the oral administration of encapsulated bacterial cells (6, 7). Similarly, it was shown that oral administration of activated charcoal can be used to minimize epithelial tight junction damage and reduce oxidative stress and inflammation in chronic kidney disease animals (8). This article combines previous oral therapies and for the first time explores the use of co-encapsulated live bacterial cells and activated charcoal to remove renal failure metabolites.
Building on research done in the late 1990s to create semipermeable membranes capable of housing live bacterial cells (17), the use of probiotic microcapsules has grown massively. From reducing cholesterol levels (18), to disrupting the pathogenesis of hepatic encephalopathy (19), probiotics’ effects on the gut microbiota cannot be underestimated. Encapsulating bacteria also allows for the mixing of different bacterial species creating novel probiotic blends. Different blends have been shown to target risk factors of metabolic syndrome (20) and Alzheimer’s (21) in-vivo and in drosophila melanogaster respectively. All of this, in addition to previous research showing that probiotics reduce other uremic toxins, suggests the importance of probiotics both as an emerging field of research and as a potential therapy for renal failure.
Using oral microcapsules to remove uremic toxins is feasible due to the presence of these toxins in plasma, suggesting the exchange of uremic metabolites between the lumen of the GI tract and its capillaries (9). This exchange was well-characterized in the mid-1900s and prompted researchers to experiment on the feasibility of intestinal perfusion as a means of removing uremic toxins to manage renal failure (10, 11). However, as intestinal perfusion procedures need to be done at frequent intervals to prevent the build-up of uremic toxins such as creatinine, they are both expensive and lower the quality of life of patients. Another alternative is to orally administer encapsulated indigestible adsorbents for the removal of uremic toxins from the GI tract (12, 13). Such adsorbents include: activated charcoal as well as various carbon and starch nanoparticles (13, 14). Activated charcoal, in the concentrations used in this experiment, is a well-established nontoxic adsorbent for creatinine and uric acid (15, 16).
The aim of this study is to co-encapsulate both activated charcoal and metabolically induced L. acidophilus 314 cells and examine their efficacy of creatinine removal in in-vitro conditions simulating those of the small intestine. Ideally this co-encapsulation would provide a model for the removal of other unwanted renal failure metabolites.
Materials and Methods
Sodium alginate (low viscosity), creatinine and urea were purchased from Sigma-Aldrich, USA. Chitosan (low viscosity) was obtained from Wako Chemicals, Japan. Activated charcoal (Norit E Supra USP) was obtained from Norit Americas Inc., USA. Ox gall (dehydrated Fresh Bile) was obtained from Difco, USA, and pancreatin purchased from Acros Organics, USA. All other chemicals were of analytical reagent grade and not purified further before use.
Metabolic induction of L. acidophilus
Lactobacillus acidophilus 314 was obtained from ATCC. Bacterial cultures were maintained in Lactobacilli MRS Broth (Difco, USA) overnight at 37 ˚C in anaerobic conditions. L. acidophilus was cultivated in 1.5 ml MRS broth, with urea levels starting from 50 mg/Dl, and increasing in increments of 0.3 g/Dl to 2.7 g/Dl. The urea solution was filtered through 0.22 μm filter and added to autoclaved MRS broth. At each urea increment, the bacterium was cultivated for several passages for adaptation, before screening for healthy colonies on modified MRS agar plates enriched with a 0.3 g/dl higher urea concentration. The colonies were then re-inoculated into MRS broth at the higher urea concentration of the modified agar plate. This process was repeated until 2.7 g/Dl urea concentration (corresponding to 120 days), after which further increases in urea resulted in a bacterial growth too scant for practical use. The induced L. acidophilus was re-adapted to a lower urea level – 150 mg/Dl – similar to pathological levels in renal failure patients and used for in vitro experiments.
Chitosan-alginate microcapsules containing bacterial cells and/or activated charcoal were prepared based on microencapsulation procedures described in a previous report (22). L. acidophilus was grown in 150 mg/Dl urea-enriched MRS solution, harvested at late log phase, and collected by centrifugation at 10 000 g for 20 minutes at 4 ˚C. The media solution was discarded, and the cell mass washed three times with physiological solution (0.85 % (w/v) sodium chloride). 1.65 % (w/v) sodium alginate in physiological solution (PS) was sterile filtered through a 0.22 μm filter. 0.9 g L. acidophilus cells were re-suspended in alginate/physiological (PS) (9:1, v/v) reaching a final volume of 30.0 Ml. The bacterial suspension was extruded through a 600 μm diameter nozzle with pressurized nitrogen using an INOTECH encapsulator that was adjusted to a frequency of 450 Hz, a voltage of 0.25 Kv and an output pressure of 0.2 bar. Minute irregularities in the air stream resulted in the formation of two different sizes of microcapsules: 99 % egg shaped or oblate ellipsoidal microcapsules and 1 % larger, spherical microcapsules. The average diameters (minor diameters) of the 99 % microcapsule population are as follows: empty microcapsules: 619 μm ± 36 μm, L. acidophilus microcapsules: 628 μm ± 24 μm, activated charcoal microcapsules: 629 μm ± 28 μm and microcapsules containing both L. acidophilus and activated charcoal: 640 μm ± 23 μm, n=30. Fig. 1 contains photomicrographs of the microcapsules under a light microscope. The droplets were stirred gently in chilled calcium chloride solution, 1.0 M (w/v), for 30 minutes and allowed to gel at 4 °C for 2 hours. Chitosan-alginate microcapsules were prepared by immersing the alginate droplets in a 0.5 % (w/v) chitosan solution in 0.5 % acetic acid (w/w) that is pH adjusted to 4.6, for 30 minutes. Microcapsules formed were washed and stored in PS at 4 ˚C until further use.
Combination microcapsules containing bacterial cells and activated charcoal were prepared according to the procedure described above, except that activated charcoal was added to the bacterial-alginate mixture prior to droplet extrusion. To test the reaction of the microcapsules in a simulated GI medium containing creatinine, the test groups include two negative controls: a) without microcapsules and b) empty microcapsules containing neither bacteria nor activated charcoal, and three test groups: c) L. acidophilus microcapsules, d) activated charcoal microcapsules and e) combination microcapsules containing both L. acidophilus and activated charcoal. Across the test groups containing the bacterial species, L. acidophilus wasstandardized in the microcapsules at 1.1 x 109 CFU/mL (colony forming units/mL) and 1.2 x 109 CFU/mL for the studies at pH 6.4 and pH 7.8 (to simulate conditions in the proximal and distal intestine respectively); while encapsulated activated charcoal was standardized at 54 mg for the studies at both pHs. Depending on the contents of the alginate mixture, the encapsulator settings differ for the extrusion of droplets and are summarized in Table 1.
InoTech Encapsulator settings for the encapsulation of a) empty microcapsules, b) activated charcoal microcapsules, c) Lactobacillus acidophilus microcapsules, d) microcapsules containing both activated charcoal and Lactobacillus acidophilus.
|Empty microcapsule||Microcapsule containing activated charcoal||L. acidophilus 314||L. acidophilus 314 & activated charcoal|
|Output pressure (bar)||0.3||0.25||0.2||0.25|
|Drop Height (inch)a||2.0||1.0||1.5||1.0|
In-vitro microcapsule studies in simulated gastric reaction media
Microcapsules were immersed in simulated GI media (consists of a carbohydrate-based diet with 1.0 g arabinogalactan/L, 2.0 g pectin/L, 1.0 g xylan/L, 3.0 g starch/L, 0.4 g glucose/L, 3.0 g yeast extract/L, 1.0 g peptone/L, 4.0 g mucin/L and 0.5 g cysteine/L. Pancreatic juice (containing 6.0 g ox gall/L, 0.9 g pancreatin/L and 12.0 g sodium bicarbonate/L) is added at 27.8 mL/100.0 mL simulated food mixture and is pH adjusted to simulate different sections of the small intestine. 12.5 mL creatinine is added per 100.0 mL simulated GI media. The microcapsules in each test group were immersed in 10.0 mL of the simulated GI media and incubated at 37 ˚C and anaerobic conditions for 48 hours. 1.0 mL of the synthetic media was sampled at 0, 10, 16, 24, 36 and 48 hours. The samples were centrifuged at 10 000 g, 4 ˚C for 10 minutes to obtain the supernatant for further analysis. Creatinine concentrations were determined using the 911 Hitachi Blood Chemistry Analyzer.
Bacterial viability in storage conditions
Using the microcapsules immediately after its production as in this study is not always feasible, therefore it is important to test for bacterial viability in storage conditions, specifically in media at 4 ºC. This study involved two test groups: L. acidophilus microcapsules and the combination microcapsules containing L. acidophilus and activated charcoal. 4 g of microcapsules was immersed in 18 mL storage medium, which was either a) 10 % MRS medium enriched with 150 mg/dL urea 90 % PS solution or b) PS solution. 0.1 mL of microcapsules was sacrificed and plated at various dilutions to determine CFU counts in 1.0 mL at 0, 1, 2, 4, and 8 weeks.
Results and Discussion
The well-established adsorbent properties of activated charcoal and the inductive potential of bacteria have motivated their co-encapsulation for the removal of the uremic toxin creatinine in the GI tract. In vitro studies were focused on two pHs (pH 6.4 and 7.8) representative of the proximal and distal sections of the small intestine. Since exchange of molecules between blood capillaries and the GI cavity is the highest in the small intestine, the encapsulated bioreactors are likely to be most effective within this section at the pHs of 6.4 and 7.8. To visualize the efficacy of the microcapsules in decreasing creatinine, values are displayed in terms of the number of moles of creatinine removed per gram microcapsules were calculated and are shown in Table 2.
Calculated valuesb showing moles of creatinine removed per gram microcapsules
|Microcapsule groups||Creatinine removed (per gram microcapsules)|
|Ph 6.4 (μmol/g)||Ph 7.8 (μmol/g)|
|Activated charcoal microcapsules||1.26||1.78|
|L. acidophilus 314 microcapsules||0.71||0.83|
|L. acidophilus 314 & activated charcoal microcapsules||0.97||1.40|
Effect of capsules on creatinine exposure at pH 6.4 (proximal section of small intestine)
After incubating the microcapsules in a simulated GI media of the proximal and distal sections of the small intestine for 48 hours in anaerobic conditions, it was shown that L. acidophilus 314 microcapsules removed 204 μmol/L creatinine compared to creatinine removal of 103 μmol/L by empty microcapsules. This suggests that L. acidophilus 314 may be able to metabolize creatinine, and that the empty capsules were able to trap creatinine following its diffusion into the alginate matrix, thus ensuring the complete removal of creatinine from the GI tract in fecal excretion after 24 hours. The largest removal of creatinine was achieved by activated charcoal microcapsules, where 379 μmol/L creatinine was removed in the same period.
From Fig. 2, the combination of L. acidophilus 314 and activated charcoal is not a superposition of their individual effects, where only 309 μmol/L ccreatinine was removed compared to 379 μmol/L by activated charcoal microcapsules. This may be due to physical obstruction of the pores on activated charcoal particles by L. acidophilus 314, resulting in the inability to utilize the full capacity of the adsorption area.
From Table 2 shown earlier, the creatinine removal capacities of the microcapsules are compared per gram microcapsules, and these values support the discussion above. Activated charcoal microcapsules removed creatinine at 1.26 μmol/g, and L. acidophilus 314 microcapsules removed creatinine at 0.71 μmol/g. The combination microcapsules showed a creatinine removal at 0.97 μmol/g. This decreased creatinine removal capacity in the combination microcapsule is consistent with the results obtained from Fig. 2 and may be due to obstruction of the activated charcoal pores by the probiotic.
It is important to consider that the decrease in creatinine seen at pH 6.4 contradicts data from similar studies, which concluded that creatinine concentrations are unaffected by probiotic treatment (23, 24, 25). However, those studies were done in-vivo with chronic kidney disease patients and with a combination of probiotic strains. On the other hand, this study was done in-vitro while isolating different regions of the small intestine and with a specific strain of bacteria. This suggests potential interplay both between the different regions of the small intestine as well as between different species of probiotic bacteria that may influence L. acidophilus 314’s overall ability to metabolize creatinine. However, more research needs to be conducted on L. acidophilus 314’s creatinine metabolic pathway and its potential influences before drawing conclusions.
Effect of microcapsules on creatinine exposure at pH 7.8 (distal section of the small intestine)
When the experiment was repeated at pH 7.8, L. acidophilus 314 microcapsules did not display the same ability to remove creatinine as was observed at pH 6.4; 250 μmol/L creatinine was removed after 48 hours, which was comparable to the 253 μmol/L creatinine removed by the empty microcapsules in the same period. This is because the pH range for L. acidophilus 314 survival is 4.0 – 6.4, therefore the slight basicity at pH 7.8 may have ceased L. acidophilus 314 activity. As was observed at pH 6.4, activated charcoal microcapsules showed the largest creatinine decrease of 533 μmol/L. This suggests that activated charcoal’s affinity for creatinine is not pH dependent within this range. Physiologically this means that activated charcoal will function throughout the entire length of the small intestine and is only limited by saturation effects.
From Fig. 3, the combination microcapsules removed 438 μmol/L creatinine compared to 533 μmol/L removal by activated charcoal microcapsules despite similar amounts of activated charcoal used for both groups. This is consistent with results obtained at pH 6.4 and further suggests the physical obstruction of activated charcoal pores by the bacteria. Since this is a physical process the reduced efficacy of the combination group is not affected by pH and is observed in both regions of the SI to similar extents. The exact mechanism of the interaction between activated charcoal and L. acidophilus 314, as well as the metabolic process of creatinine uptake by the L. acidophilus 314 cells is not fully understood and further research in this area is needed.
A comparison of the creatinine removal capacities can be made from Table 2 and supports the discussion above. Creatinine was removed at the capacities of 1.78 μmol/g activated charcoal microcapsules and 0.83 μmol/g L. acidophilus 314 microcapsules. The creatinine removal capacity between L. acidophilus 314 microcapsules and empty microcapsules (0.84 μmol/g) were comparable, indicating that L. acidophilus 314 did not remove creatinine at pH 7.8. Similar to pH 6.4, the combination microcapsules removed creatinine at a lower capacity compared to activated charcoal microcapsules, at 1.40 μmol/g combination microcapsules to 1.78 μmol/g activated charcoal microcapsule.
Comparing the creatinine removal capacity of 1 g microcapsules from the same test group between the two pHs, a higher removal capacity was observed at pH 7.8. This phenomenon is observed across the four microcapsule test groups. This may be due to changes in the properties of the alginate core or the chitosan membrane of the microcapsule. However, more studies are needed to examine this change in properties at different pHs.
Effect of activated charcoal on bacteria viability
The addition of activated charcoal to the bacteria culture did not negatively affect bacterial viability. Both L. acidophilus capsules and co-encapsulated capsules contained 109 CFU/ Ml at the start of the experiment. At the end of 48 hours, microcapsules containing activated charcoal and L. acidophilus decreased CFU counts by one order of magnitude, while L. acidophilus microcapsules showed a decrease of two orders of magnitude (data not shown).
Acid tolerance test on bacteria viability
Initially, when placed in pH 1.9 acidic media simulating the GIT, both L. acidophilus 314 microcapsules and co-encapsulated microcapsules showed similar CFU counts. However, at the end of 120 minutes, bacterial CFU decreased 1 ½ orders of magnitude in the combination capsule, compared to a reduction of about two orders of magnitude in the L. acidophilus 314 capsule (Fig. 4). This small but significant difference suggests supportive functions undertaken by activated charcoal to preserve bacterial viability. Depending on food intake, the microcapsules may transit in the stomach for a maximum of 2 hours, and these results indicate the rate of decrease of bacterial viability which will help determine time and dosage administration. Although creatinine removal by the combination capsules was less effective than activated charcoal capsules, the combination capsule may be the superior choice as a result of the supportive functions of activated charcoal on the bacterial cells.
Bacterial viability in storage conditions
Results show that microcapsules containing activated charcoal preserved bacterial viability at least 5 weeks longer than microcapsules containing only bacteria; L. acidophilus microcapsules showed 0% bacterial viability at week 3. In contrast, the combination microcapsules showed bacterial viability until the end of week 8 when the experiment was concluded. This supports the observation that activated charcoal provides support for bacterial viability.
Upon comparison of the two storage media it is observed that the PS storage resulted in higher bacterial viability compared to the storage in 10 % urea enriched MRS broth. Comparing only L. acidophilus microcapsules, the CFU count for the microcapsules stored in PS was 6.7 x 108 CFU/mL at week 2 compared to the 1.1 x 107 CFU/mL CFU count for the microcapsules stored in 10 % urea enriched MRS broth. Further comparison of the combination microcapsules show that those stored in PS reflected a slower decrease of bacterial CFU counts than those in urea enriched broth; the combination microcapsules stored in PS reported a CFU count of 1.0 x107 CFU/mL compared to a CFU count of 2.0 x 103 CFU/mL for those stored in 10 % urea enriched MRS medium. This shows that the additional urea may have caused stress to the survival of the bacteria at low temperatures. As a side note, MRS broth without urea was not tested as a storage medium because of the possibility of compromising the inductive capacities of the bacteria.
The efficacy of creatinine removal by microcapsules containing L. acidophilus 314 and activated charcoal was evaluated in this study. Results show that combination microcapsules were not as efficient as the microcapsules containing activated charcoal only. However, activated charcoal in the combination microcapsule helped preserve bacterial viability during transit in media simulating the stomach, suggesting an advantage to the combination microcapsule despite its reduced efficacy. The addition of activated charcoal also improved the bacterial viability of microcapsules, allowing them to be stored for longer and survive better in the GIT. However, due to the inconsistencies in creatinine uptake of L. acidophilus in the different pH environments, as well as conclusions made by other studies, more research is needed to understand creatinine’s metabolic pathway in L. acidophilus. Regardless, this study may provide a model for the metabolic induction of different bacteria strains for the uptake of various unwanted metabolites. Microcapsules containing activated charcoal and probiotic bacteria may also potentially serve as an oral adjuvant to reduce the frequency and duration of dialysis; however, further research is still required.
This work was Natural Science and Engineering Research (NSERC), Canada. Ouyang received postdoctoral fellowship from Fonds Québécois de la Recherche sur la Nature et les Technologies (FQRNT) and Lim received postgraduate scholarship from Natural Science and Engineering Research (NSERC), Canada.
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The height refers to the drop height measured from the nozzle tip to the surface of the calcium chloride solution.
The calculation is as follows: The number of moles of creatinine in the 10 Ml reaction media is first calculated by the equation: Y moles/1000 Ml x 10 Ml = y moles. The number of moles of creatinine at the end of the 48-hour experiment is also calculated (x moles). Their difference gives the total amount of creatinine reduced ((y – x) moles). When divided by the wet weight of microcapsules used, this yields the moles of creatinine removed per gram microcapsule.