*4.5. Also, Other Bilirubin Related Blood Biomarkers Indicate Strong Hepatic Activation in Four Days*

Blood Fe declined strongly in the LC subgroup during the 4-day DGA regimen. Furthermore, there was a very strong association (*p* < 0.0001) between the relative changes in bilirubin and Fe in blood. These two events indicate that hepatic iron demand was also increased [38]. However, it should be noticed that also HO-1 pathway downregulation could explain, at least partly, the strong correlation between the changes in bilirubin and Fe [1]. The strong 16.3% reduction in Fe in the LC subgroup may be additionally explained by the mitochondrial activation during the 4-day DGA regimen [27]. Mitochondrial activation increases the use of Fe in OXPHOS heme proteins [33]. This is because the liver especially seems to be activated significantly during the 4-day DGA regimen [27], and the demand of blood Fe for mitochondrial heme protein synthesis may have increased the most in the liver [40]. The blood Fe pool is only 3–4 mg and must turn over several times daily to meet the demand, mostly from erythropoiesis [33]. All in all, hepatic activation could explain most of the strong decline in blood Fe during the 4-day DGA regimen.

The four-day changes in blood TGs were also associated with the changes in Fe (R<sup>2</sup> = 0.446, Figure 5C). This surprisingly strong relationship may indicate that the hepatic activation [41] in the LC subgroup may also be behind the decline in blood TGs in the LC subgroup. Change in blood TGs concentration is directly related to the net release of TGs from the liver [42], and a more active liver consumes a bigger part of formed TGs itself [43].

Finally, unconjugated bilirubin presumably flows into the liver with albumin and at the same time also the HO pathway was likely activated in the liver. To avoid excessive concentration of potentially toxic bilirubin, it is likely that hepatic conjugation and excretion of the conjugated bilirubin increased simultaneously. Conjugated bilirubin is excreted from hepatocytes as bile pigment via the bile ducts. ALP may be increased even if only a few small bile ducts are obstructed, and serum bilirubin is normal [44,45]. Remarkably, plasma ALP was reduced in each LC participant during the 4-day DGA activation (*p* < 0.0001). The extremely consistent reduction in ALP and the reduction in blood bilirubin in the LC subgroup may point to some unspecified hepatobiliary enhancement, such as the bile acid secretion to the small intestine [3], during the 4-day DGA regimen in the LC subgroup.

#### *4.6. In Four Days, Systemic Inflammation (GlycA) and TGs Correlated Inversely with Blood Bilirubin*

The 21-day changes in blood bilirubin and GlycA correlated negatively. During the 4-day DGA regimen, correlation between the changes in bilirubin and GlycA was positive, i.e., totally opposite to that on Day21. A technical explanation for this inverse 4-day bilirubin–GlycA correlation arises from the strong reduction of systemic inflammation in the LC subgroup and simultaneous modest increase in the HC subgroup, and from similar subgroup dynamics in blood bilirubin.

What happens during the 4-day DGA regimen that temporarily distorts the canonical relationship that has been observed also in the literature [1,23,24]? We do not know the exact reason, but the hepatic activation that is accompanied with the reduction in systemic low-grade inflammation may be an important explanatory factor during the 4-day DGA regimen. Additionally, it is possible that in the LC subgroup, the HO-1 pathway activity was reduced due to reduction in inflammation, and as a follow-up, the release of bilirubin and Fe from tissues to blood was reduced causing the inverse correlation. All in all, the positive 4-day correlations between GlycA and bilirubin, and GlycA and Fe can be likely explained by the activation of the liver and the downregulation of whole-body HO-1 pathway activity due to reduction of systemic inflammation.

Also, the correlation between the changes in bilirubin and TGs during the 4-day DGA regimen was positive, i.e., fully the opposite than during the period from Day0 to Day21. Partial explanation for the positive correlation likely arises from the presumed inflow of bilirubin into the liver in the LC subgroup. It possibly increased hepatic fatty acid oxidation via PPARα [8] and tended to reduce blood TGs more in participants with higher hepatic bilirubin inflow from blood, and vice versa. Further explanation for the positive correlation between bilirubin and TGs during the 4-day DGA regimen arises from the strong correlation between the changes in TGs and GlycA during the same 4-day period (R2 = 0.686), i.e., the correlations between the changes in blood TGs, bilirubin, and Fe were partially due to very strong association between the changes in GlycA and TGs.
