Second Place: The Effects of Oxazyme on Oxalate Degradation: Results and Implications of In Vitro Experiments

Introduction

O xalate is the dianion conjugate base of oxalic acid, a dicarboxylic acid, and is a metabolic end product in humans. It is excreted in the urine and influences the development of calcium oxalate kidney stones, the most common stone composition in the United States. ^1,2 Approximately 50% to 60% of urinary oxalate originates from dietary sources, while 40% to 50% is derived from endogenous synthesis thought to occur in the liver. ³

A goal for calcium oxalate stone formers, especially those who are hyperoxaluric, is to reduce urinary oxalate excretion. Initial steps include a reduction in oxalate consumption and maintenance of normal dietary calcium intake. This theoretically results in reduced soluble complexes of calcium and oxalate, limiting the gastrointestinal absorption of oxalate and its delivery to the kidney. ⁴ Probiotic bacterial preparations containing oxalate-degrading organisms have been administered to patients in an effort to reduce urinary oxalate excretion. Some have reported a reduction in oxalate excretion, while others have not found this response. ^5,6

Oxalate decarboxylase is an enzyme that catalyzes the metabolism of oxalate to carbon dioxide and formate. If this enzyme was consumed by humans, it could theoretically limit the oxalate pool in the gastrointestinal tract, resulting in reduced urinary excretion. Oxazyme® (Oxthera, Alachua, FL) is an oral formulation of recombinant mutant oxalate decarboxylase derived from Bacillus subtilis that functions optimally at a pH range of 3 to 4. In 2010, Cowley and colleagues ⁷ investigated its toxicity in 14-day, repeated dose studies performed in dog and rat subjects. No adverse effects were noted in either species at or below the following maximum doses: 720.8 mg/kg/day in rats and 187.2 mg/kg/day in dogs.

Oxalate is absorbed throughout the gastrointestinal tract with a significant amount occurring in the small intestine. ⁸ Furthermore, soluble oxalate is more readily absorbed than insoluble or crystalline oxalate. We undertook this study to determine whether Oxazyme could degrade oxalate in an environment simulating the gastric lumen where its activity would be optimized and the small intestine where pH is substantially higher. This should help predict whether this enzyme preparation will have clinical utility in the future.

Materials and Methods

We utilized powdered Oxazyme^®, provided directly from Oxthera laboratories in sachets. This preparation also contains mannitol. In this form, Oxazyme is maintained within microencapsulated molecules containing the following: Trehalose, triethyl citrate, and Eudragit L100-55. The first two components have received GRAS designation from the GRAS Expert Panel, and the last is a common copolymer found in many approved drugs.

Because Oxazyme optimally functions at a pH of 3 to 4, which is similar to the pH of stomach fluid after the ingestion of a meal, we performed experiments in a simulated postprandial gastric environment. We also investigated Oxazyme's function in a simulated proximal intestinal environment where the pH is approximately 6 to 6.5. A pH-specific, biorelevant media similar to those previously described by Jantradid and coworkers ⁹ was used. This resulted in a simulated gastric media with a pH of 3.6 and a simulated small intestinal milieu with a pH of 6.5.

Oxalate sources used included powdered potassium oxalate and spinach; the latter, which is readily available, has a known oxalate content and is one of the most common oxalate-rich foods (770–795 mg per 100 g). ¹⁰

Samples of spinach and potassium oxalate were assessed with and without the presence of Oxazyme in both the simulated intestinal and gastric buffers. For samples containing spinach, 1 g of the leafy green vegetable was added to 25 mL of buffer. The spinach was prepared in two ways to assess the impact of this on the exposure to Oxazyme: Whole leaf spinach and spinach homogenized using a Polytron blade homogenizer (Capitol Scientific, Inc., Austin, TX) for 30 seconds.

Regarding the potassium oxalate specimens, 0.5 mL of a 100 mM potassium oxalate preparation was combined with 25 mL of the gastric or intestinal buffer. Samples were prepared in duplicate to allow one of the samples to serve as a “control” while the other sample contained 10 mg of Oxazyme. Each pair of samples was then incubated in the appropriate buffer on a laboratory rocker for 1 hour at 37°C. Each of these paired incubations was repeated four times. While the samples incubated, Nano-sep centrifugal filters (VWR International, Batavia, IL), with a 10,000 nominal molecular weight limit, were prepared by first centrifuging 300 μL of 0.1 M HCL at 13.8 relative centrifugal force (RCF) for 6 minutes, followed by 300 μL of deionized water at 13.8 RCF for 6 minutes. This was performed to remove any oxalate in these filters. Subsequently, 200 μL of the sample was added and centrifuged at 13.8 RCF for 10 minutes. These samples represented the homogenized spinach, the potassium oxalate mixture, and, for the whole leaf specimens, only the liquid around the leaf. Samples were diluted 50- to 100-fold in sodium acetate for analysis, with the extent of the dilution depending on the oxalate and anion contents of the sample.

The oxalate content of our samples was determined using ion chromatography (IC) (Thermo Fisher Scientific, Inc, Waltham, MA), with suppressed conductivity detection at 10 mA using an AS-22, 2×250 mm ion exchange column and a 2.5 mM sodium carbonate/1.7 mM sodium bicarbonate buffer at 0.3 mL/min. The oxalate peak elutes at 22 to 24 minutes. The accuracy and reproducibility of IC in estimating the oxalate content of foods has previously been demonstrated. ¹⁰ We also tested the buffers (with and without Oxazyme) for oxalate content.

All chemicals were of analytical grade and were purchased from Sigma-Aldrich (St. Louis, MO). Spinach was obtained fresh at a local emporium.

Statistical analysis was performed using the Student t test.

Results

Oxazyme resulted in the substantial reduction of oxalate derived from potassium oxalate when incubated in either of the simulated buffers. In fact, in the intestinal buffer, oxalate was completely eliminated by the addition of Oxazyme.

Similarly, a profound decrease in the concentration of oxalate was demonstrated when Oxazyme was exposed to whole leaf and homogenized spinach in the gastric or intestinal simulated buffers. Oxalate was not detected in either buffer or Oxazyme (Table 1).

Discussion

Oxalate decarboxylase is a manganese-containing, multimeric enzyme belonging to the cupin protein superfamily. ¹¹ It is one of three enzymes that have been identified with the ability to decompose oxalate and oxalic acid. The other two enzymes are oxalate oxidase, mainly found intracellularly in plants, and oxalyl-CoA decarboxylase, a bacterial enzyme notably found in Oxalobacter formigenes. ¹² Previously used in beer brewing processes, biotechnologic applications, and in agriculture, the administration of oxalate decarboxylase is currently being considered for reducing urinary oxalate excretion in humans. ^13,14 Originally described in the mycelial extracts of the fungi Trametes hirsuta and Flammulina velutipes, oxalate decarboxylase has since been detected in bacterial species, animal tissue, and other fungi. ^15,16

The most extensively investigated form of oxalate decarboxylase is that which is isolated from the bacterium B subtilis. The expression of this enzyme in the cell wall of B subtilis is found to be profound during acidic stress conditions. ¹⁷ This bacterial enzyme has been the model for numerous studies aimed at deciphering the catalytic mechanism of oxalate decarboxylase. It is believed that the carbon-carbon bond of oxalate is cleaved by oxalate decarboxylase, producing formate and carbon dioxide, in a reaction that incorporates the shifting of electrons from manganese to oxygen. ¹⁸

Recent experimentation has focused on the use of oxalate decarboxylase as a potential mechanism to decompose intestinal oxalate in humans. After the discovery that the YrvK gene of B subtilis encodes the 43 kD oxalate decarboxylase, investigators were able to develop recombinant Escherichia coli, expressing this gene, which demonstrated potent oxalate-degrading activity when administered orally to hyperoxaluric rat models. ^19,20 Extrapolating from these findings, Grujic and associates demonstrated that oral administration of recombinant B subtilis oxalate decarboxylase to hyperoxaluric mice resulted in decreased oxalate urinary excretion and prevention of nephrocalcinosis. ²¹ Another group of scientists reported the reduction of oxalate content in tea by adding oxalate decarboxylase during the brewing process. ²²

Within the last 5 years, Oxazyme, an orally administered formulation of oxalate decarboxylase, has been developed as a potential therapeutic agent for reducing urinary oxalate excretion. To date, no human studies evaluating the safety and efficacy of Oxazyme have been reported.

We undertook this study to determine whether Oxazyme could degrade oxalate in simulated gastric and small intestinal environments. These are areas where food-derived oxalate would initially be delivered to the gastrointestinal tract. Our results clearly demonstrate that this compound is quite effective in degrading oxalate in such environments. This is expected in the gastric environment, because the pH is within the range for its optimal activity. A significant amount of oxalate is also absorbed from the small intestinal compartment. ⁸ The results of this study also suggest that even in an environment where the pH is out of the optimal range of activity (intestinal buffer), Oxazyme may prove to be effective in metabolizing oxalate.

We recognize that this study has certain limitations. The long period of incubation could have contributed to the near complete degradation of oxalate in the presence of Oxazyme. In addition, known gastric enzymes, such as pepsin, and pancreatic enzymes, were not added to the respective media. This would have better simulated the gastric and intestinal environments. In vitro studies are not always predictive of in vivo responses. In addition, other high oxalate containing foods were not assessed, and the impact of other foods consumed at the same time may alter results. There may be substantial variability in the amount of oxalate within spinach and between batches of this plant. This has previously been observed not only in spinach, but in other foods, such as sweet potatoes. ¹⁰ Factors known to influence the oxalate content of plants include the plant variety, the developmental stage of the plant, the season, and growth conditions. ²³ The possibility that our spinach specimens varied significantly in their oxalate content could have impacted the specific data, but not the overall, consistent display of Oxazyme's ability to metabolize oxalate.

Conclusion

Our results suggest that Oxazyme appears to have the potential to metabolize oxalate in simulated gastric and small intestinal environments.