Butyrate and clinoptilolite

Amazon sells a clinoptilolite as a garden soil supplement. The material is fairly inexpensive but will need to be ground down and calcinated to remove organic material. The featured study relates to two previous posts that will be noted in this one. I found this paper extremely confusing with the biggest asset being (1) the protocol as to how to make, and (2) suggestion of inducing glutathione peroxidase activity and/or mimetic activity. Other changes were statistically significant but not that dramatic.

Wu Y, Zhou Y, Lu C, Ahmad H, Zhang H, He J, Zhang L, Wang T. Influence of Butyrate Loaded Clinoptilolite Dietary Supplementation on Growth Performance, Development of Intestine and Antioxidant Capacity in Broiler Chickens. PLoS One. 2016 Apr 22;11(4):e0154410. PMC free article

This studly used 240 1-d-old Arbor Acres broilers from a local hatchery in China. The experimental designed consisted of four groups with six replicas containing 10 birds each. Each treatment had six replicates. Each replicate contained ten birds with similar average body weight and were fed soy and corn diets whose nutrients were age adjusted and the chicks grew.

• as basic diet (CON group)
• basic diet + 0.05% sodium butyrate (SB group),
• basic diet + 1% clinoptilolite (CLI group),
• basic diet + 1% butyrate loaded clinoptilolite (CLI-B group).

Preparation of clinoptilolite and butyrate loading

• sieved through a 100-mesh sieve
• calcined in a muffle oven at 350°C for 2 h to remove the water and organic template and to free the pores.
• Butyrate (Chemical Pure, 98%) and sodium butyrate (Chemical Pure, 98%) The clinoptilolite was added to a 70-mL butyrate solution with a concentration of 3 mol/L.
• The mixture was blended at 60°C at 151 rpm/min in a constant temperature oscillated instrument
• for 4 h, and the lower sediments were washed by deionized water until the pH of the washed
• solution was 7.
• Finally, the washed material was collected and dried at 105°C for 2 h in an air oven and then ground and sieved through a 100-mesh sieve. The temperature (105°C) was much lower than the boiling point of butyrate (163.5°C).  This part of the protocol was thought to remove water as well.   

The butyrate loaded into the clinoptilolite was 3.8%.,

Tables 1 &2 food conversion ratio and pancreatic lipase

The body weight gain and food intake are not that different. There seems to be an improvement n the food conversion ratio, or the kg of food consumed to gain a kg of mass of body weight for the Clinoptilolite-Butyrate prep compared to the control. This seems to be more pronounced at the 21-42 and 1-42 day time periods. For some reason, pancreatic lipase is more improved than proteases and amyases.

Tables 3-4, improvements in villus length in the duodenum

The improvements in weight and villus length might be statistically significant, but it can be argued that they might not be practically significant. Butyrate fires through GPR41 and GPR43 in the GI tract. This particular post discusses improvements in transepithelial electrical resistance elicited by butyrate in a cell culture model. It should be noted that the first clinoptilolite post described improved GI barrier function with clinoptilolite without the butyrate.

There really does not seem to be any difference in tight junctions in the set of images in the above figure.

The interesting thing about Table 4 is that the most dramatic changes are seen at day 21.

Table 5 Serum Nitric Oxide synthase activity

There are three mammalian isoforms of NOS

• nNOS, neuronal, controls synaptic plasticity and smooth muscle relaxation.
• eNOS, endothelial, controls vascular relaxation
• iNOS, deducible in cells of the immune system, controls the body’s response to pathogens.

Serum nitric oxide (NO, μmol/L), total nitric oxide synthase (TNOS, U/L) and induce nitric oxide synthase (iNOS, U/L) were measured with commercial kits according to the manufacturer’s instructions.

All treatments result in a significant decrease in serum nitric oxide, on days 21 and 42. Only butyrate treated clinoptilolite decreased iNOS activity on day 21.

Table 6, anti-oxidant enzymes in tissue homogenates

This table was very confusing. The authors were looking at three antioxidant enzymes in the jejunum and ileum. In two cases they measured enzyme activity and the amount of enzyme. This fits into the general narrative that clinoptilolite can deliver metal cofactors and butyrate can induce or turn off gene transcription. Catalase requires iron as a cofactor. This catalytic cycle is as follows:

• H₂O₂ + Fe(III)-E → H₂O + O=Fe(IV)-E(.+)
• H₂O₂ + O=Fe(IV)-E(.+) → H₂O + Fe(III)-E + O₂^[

The only difference is in the amount of catalase activity at day 21 in the jejunum. The difference is not that large. This does not support the notion that clinoptilolite is delivering the Fe cofactor.

Glutathione peroxidase uses selenium as a cofactor. It’s catalytic cycle is as follows:

• RSeH + H₂O₂ → RSeOH + H₂O
• RSeOH + GSH → GS-SeR + H₂O
• GS-SeR + GSH → GS-SG + RSeH

GSH-Px relies on selenium as a cofactor, an element with similar chemistry to sulfur right below sulfur on the Periodic Table of Elements.

• In the jejunum on day 21 butyrate-clinoptilolite improved total anti-oxidant capacity.
• In the jejunum on day 42 the butyrate-clinoptilolite combo was required to increase specific GSH-Prx activity compared to the control.
• None of the treatments made a difference in the ileum on day 21.
• By day 42 clinoptilolite with or without butyrate improved the SGH-Prx activity and total anti-oxidant capacity compared to the control or sodium butyrate alone. .

Cu/Zn superoxide dismutase (Cu/Zn SOD) relies on Cu and Zn as cofactors. The catalytic cycle is as follows:

• Cu²⁺-SOD + O⁻₂ → Cu⁺-SOD + O₂ (reduction of copper; oxidation of super oxide)
• Cu⁺-SOD + O⁻₂ + 2H⁺ → Cu²⁺-SOD + H₂O₂ (oxidation of copper; reduction of super oxide)

• None of the treatments make a difference in the jejunum on day 21.
• In the jejunum on day 42 it is the total SOD activity improved with the clinoptilolite with or without butyrate.
• Likewise, none of the treatments made a difference in the ileum on day 21.
• In the ileum on day 42 the total SOD activity is increased by clinoptilolite with or without butyrate. These increases were a little over 50%.

Parting remarks

I think that clinoptilolite-butyrate combo is promising. I question whether or not the butyrate is release from the clinoptilolite. The combo seems to be acting as an anti-inflammatory. They had some nice H&E stained images. It would have been nice to have evidence of “leaky gut.”