This investigation was conducted by the School of Veterinary Medicine at the University of Zagreb, Croatia. This study used a total of 16 high-yielding Holstein-Friesian dairy cows averaging 680 ± 30 kg of body weight, between 2–7 years of age. The cows were fed a control diet of haylage, corn silage, and a complete feed mixture with 19% of crude protein. The dietary forage-to-concentrate ratio was5:25 and 60:40
50gm of Zeolyte CPL was supplemented with feed.Zeolite CPL was modified by vibro-activation and micronization (Vibrosorb®, Viridisfarm, Podpi´can, Croatia). Cows were randomly assigned to the treatment and control groups. Treatment groups received the lay in the feed twice daily. No significant differences in milk yield were found between the groups of cows (the CLP group was 8325.5 ± 628.8 kg vs. the control group which was 8050 ± 586.8
Note that blood was drawn pre- and post-partum. Figure 2 was never adequately explained in this publication. The axis labels had to be zoomed in on in a PDF file in order to read them. An Internet image search of “sparse partial least squares discriminant analysis ” to reveals images of similar graphs with “component 1 on the X-axis and “component 2” on the Y-axis. Orthogonal in statistics generally means “uncorrelated, or linearly independent.” according to a blog by Eran Raviv. We will assume for now that the T score is the sum of all parameters that tend to be associated with the supplement group and that the orthogonal T score is the sum of those parameters that tend to be associated with the control group. Note that the “metabolomics” T scores do a really good job of separating the control and supplemented cows. In terms of protein expression in the plasma, only one control cow and owe supplement cow were indistinguishable. 
Just 50 g of clinoptilolite twice a day with feed completely separates the two groups of cows. One control and one supplement cow had similar T and orthogonal T scores for proteins. Volcano plots were used to present the comparison of metabolite and protein levels in the two pre- and post-partum time points in the control versus the clinoptilolite supplement groups.
Note that the red dots, indicating a positive fold change in going from control to clinoptilolite supplement, are slightly out numbered by blue dots indicating a decrease. Also note that the horizontal line between 0 and 2.5 is were we’d expect p= 0.05 on a long10 scale. With proteomics variables, we see mostly decreases in proteins. Also note hat these sets of four graphs correspond to the two pre- and post-partum time points.
Perhaps the theme is avoiding negative energy balance, that we will see is tapping into proteins as a source of energy. These are some descriptions from PubChem and/or Wikipedia
And again these figures are almost impossible to read. The bright green is the clinoptilolite supplement and the red the control.
- β hydroxy butyrate is a product fatty acid beta oxidation.
- 4-hydroxy-2-keto glutaric acid appears to be 2-oxo-gluturate…or is also known as α-Ketoglutaric acid, the a deamination production of the amino acid glutamic acid undergoing further oxidation, perhaps to enter the TCA cycle or fatty acid synthesis.
- D-Leuric acid is also known as 4-hydroxy caproic acid, a deamination product of the branched side chain amino acid leucine.
- valproic acid is a compound, according to PubChem, “it derives from valeric acid. A lot of effort was put into searching for a human (and bovine) pathway. The simplest explanation is that the investigators were really looking at a deamination production of isoleucine.
- Gycerol is a byproduct of fatty acid metabolism.
BHBA goes down in response to clinoptilolite. A yellow down arrow is drawn by the structure and by the position of BHBA in the branched chain amino acid metabolism. BHBA is again also a product of fatty acid metabolism. The structures of leucine and isoleucine are shown on the right.
It would be nice to see other evidence of metabolism of branched chain fatty acids such as perhaps nitrogen from deamination???
Clotting Cascade proteomics
These are their data. Expect for blood copper and iron carrier protein ceruloplasmin may have something to do with the complement/ clotting cascades. Serpins are a family of protease inhibitors that Wikipedia authors list thrombin as one potential protease. Fibronectin is a blood vessel surface protein that binds the blood clot protein fibrin.
The dose of 50 g clinoptilolite twice daily given to 680 ± 30 kg cows is certainly in the same range as that which humans take. It is hard to say what catabolism of branched chain amino acids for energy has to do with complement/clotting cascades. Yet this clay influenced both. Maity and coauthors suggested that the proteomics data are indicative of an inflammatory response. The decrease in ceruloplasmin supports this notion. Activated GI macrophage may secrete cruloplasmin according to Bakhautdin 2013. These data feed back on the original clinoptilolite report on human endurance athletes showing better intestinal barrier function.
- Maity S, Rubić I, Kuleš J, Horvatić A, Đuričić D, Samardžija M, Ljubić BB, Turk R, Gračner D, Maćešić N, Valpotić H, Mrljak V. Integrated Metabolomics and Proteomics Dynamics of Serum Samples Reveals Dietary Zeolite Clinoptilolite Supplementation Restores Energy Balance in High Yielding Dairy Cows. Metabolites. 2021 Dec 5;11(12):842. PMC free article
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