4.1.6. Urea

The urea measured in the blood of horses that had received knotweed in the previous winter and spring was higher than the urea in the blood of the control horses. All of these horses spent the extremely hot and dry summer between the two successive experiments on pastures and only fed on grass. It was reported [58] that changes in grass quality in the growth season and its protein (nitrogen) content correlate with blood urea level but do not affect the blood protein in horses on grass. The decreased grass quality resulted in lower blood urea content by up to half of its normal level [59]. In this study, the higher urea level in earlier knotweed-fed horses could indicate their better protein availability as all of the horses were on the same grass.

#### 4.1.7. Urea, ALP, Triglycerides and Cholesterol Interactions

Urea was also positively correlated with ALP decreasing with horse weight, the decrease being enhanced by knotweed in Experiment 1. In Experiment 2, ALP was negatively correlated with cholesterol (r = −0.435, *p* = 0.03) in the control horses but not in the knotweed-fed horses, revealing the role of ALP in the metabolism of phosphorus associated with cholesterol presumably as phospholipids, i.e., amphiphatic molecules enabling the water-solubility of cholesterol. Similarly, the cholesterol and triglycerides showed a non-significant correlation in the horses fed knotweed compared to the horses not fed knotweed, where a highly significant correlation was found (*p* = 0.001).

When all of the age categories were evaluated together, the same knotweed effect was noted at the end of both experiments: the control horses not fed knotweed had a highly significant correlation between triglycerides and cholesterol (*p* = 0.01 in Experiment 1 and *p* = 0.001 in Experiment 2), but in the blood of all of the knotweed-fed horses, there was a non-significant correlation between triglycerides and cholesterol in either experiment. When only 1- and 2-year-old horses from Experiment 1 were taken into account, reappearing as 2- and 3-year-old horses in Experiment 2, a significant correlation was found only in the horses not fed knotweed before the start of Experiment 2. This reveals a strong influence on this ratio of 3-year-old horses, i.e., the heaviest ones.

These urea, ALP, cholesterol and triglyceride interrelations indicate the effect of knotweed on the protein and lipidic (lipoprotein, phospholipid) metabolism of horses. Several authors [59,60] have reported largely increased triglyceride content due to the seasonal food deprivation of only grass-fed equines. Others [61,62] have discerned three major classes of lipoproteins in equine plasma: very low density lipoproteins (VLDL, 24 per cent of the total plasma lipoprotein mass) rich in triglycerides and cholesterol and poor in protein (apolipoprotein B-100 and apoB-48), cholesterol-rich low density lipoproteins (LDL, 15 per cent) with three discrete subfractions, and protein-rich HDL with the dominant protein apoA-I (HDL, 61 per cent), all composed of lipids and apoliproteins in different proportions. Mass spectrometry revealed that the apolipoproteins contain etw. 400–500 amino acids. These might serve for protein synthesis as one of the means of lipoprotein utilisation.

Several papers [59,63] report that in horse serum (plasma), the cleavage of VLDL mainly releases triglycerides, while LDL largely releases cholesterol. In this study, the loss of correlation between triglycerides and cholesterol in knotweed-fed horses as compared with control ones could be explained if different types of lipoproteins were cleaved in knotweed-fed horses than in control horses not fed knotweed.

A similar loss of (negative) correlation in knotweed-fed horses, which was recorded between cholesterol and ALP, would sugges<sup>t</sup> that, besides apolipoproteins, phospholipids as solubilising components and ALP targets are associated with the cholesterol particles.

The low quality of pasture grass later in the summer might be perceived as a period of mild fasting, producing higher blood triglyceride levels [59,60]. However, all of the changes measured in the blood plasma were within normal limits. Ref. [64] states that equine energy metabolism evolved in sparse grassland environments and has been adapted to the intake of a low energy diet with large quantities of roughage rich in cellulose and lignin, and that equids shift between the glucose-oriented metabolic pattern of non-ruminants and a metabolism similar to that of the ruminants when fed with a roughage-based diet. Since the blood glucose level is maintained within a narrow limit, preservation of its supply depends on gluconeogenesis, and protein catabolism increases to provide amin acids as precursors for glucose synthesis. However, more detailed studies are needed to reveal which metabolic processes are affected by knotweed in horse diets.

Meanwhile, we assume that even in earlier knotweed-fed horses, the higher urea levels indicating higher protein availability, together with a change in the ratio of lipidic substances, might have resulted from the long-lasting effects of knotweed on the metabolism of

lipoproteins. Different types of lipoproteins would be metabolised with varying intensities, and the apolipoproteins released, together with other internal sources, might help horses overcome the periods of protein starvation on dry pastures lacking high quality grass.
