**Feeding Formula Eliminates the Necessity of Bacterial Dysbiosis and Induces Inflammation and Injury in the Paneth Cell Disruption Murine NEC Model in an Osmolality-Dependent Manner**

#### **Shiloh R Lueschow 1, Stacy L Kern 2, Huiyu Gong 2, Justin L Grobe 3, Je**ff**rey L Segar 4, Susan J Carlson 2 and Steven J McElroy 1,2,\***

Received: 26 February 2020; Accepted: 24 March 2020; Published: 26 March 2020

**Abstract:** Necrotizing enterocolitis (NEC) remains a significant cause of morbidity and mortality in preterm infants. Formula feeding is a risk factor for NEC and osmolality, which is increased by the fortification that is required for adequate growth of the infant, has been suggested as a potential cause. Our laboratory has shown that Paneth cell disruption followed by induction of dysbiosis can induce NEC-like pathology in the absence of feeds. We hypothesized adding formula feeds to the model would exacerbate intestinal injury and inflammation in an osmolality-dependent manner. NEC-like injury was induced in 14–16 day-old C57Bl/6J mice by Paneth cell disruption with dithizone or diphtheria toxin, followed by feeding rodent milk substitute with varying osmolality (250–1491 mOsm/kg H2O). Animal weight, serum cytokines and osmolality, small intestinal injury, and cecal microbial composition were quantified. Paneth cell-disrupted mice fed formula had significant NEC scores compared to controls and no longer required induction of bacterial dysbiosis. Significant increases in serum inflammatory markers, small intestinal damage, and overall mortality were osmolality-dependent and not related to microbial changes. Overall, formula feeding in combination with Paneth cell disruption induced NEC-like injury in an osmolality-dependent manner, emphasizing the importance of vigilance in designing preterm infant feeds.

**Keywords:** necrotizing enterocolitis; immature intestine; formula; osmolality; inflammation; microbiome

#### **1. Introduction**

Necrotizing enterocolitis (NEC) remains the leading cause of gastrointestinal morbidity and mortality of premature infants, leading to the death of 1/3 to 1 2 of the infants who develop disease [1,2]. Although the pathophysiology of NEC is still unknown, it is postulated to be the result of bacterial translocation across the immature epithelial barrier leading to tissue invasion, subsequent inflammation, and ultimately destruction [3,4]. In general, NEC onset is preceded by a bloom in Enterobacteriaceae species, although there has been no specific microorganism determined to be causative [5–11]. Studies have linked bacterial dysbiosis or microbiome disruption with NEC development and many murine NEC models capitalize on this fact by inducing bacterial dysbiosis, disruption of the microbiome, or utilizing lipopolysaccharide (LPS), particularly in young mice [12–16]. Formula feeding has been associated with increased risk for development of NEC [17] and in rat models has been shown to

induce an inflammatory response in the immature intestine [18]. As an inflammatory milieu can induce dysbiosis in the immature intestine [6], it is reasonable to think that formula-induced inflammation may be one cause of the dysbiosis seen prior to development of NEC.

Our prior work has shown that NEC-like injury can be induced in 14–16 day-old mice (intestinally equivalent to a 22–24 week infant [19,20]) by disrupting Paneth cell biology followed by creation of an intestinal bacterial dysbiosis [21–24]. This model has direct relevance to human biology as multiple investigators have found a decrease in Paneth cells or their granular components following development of NEC [25–28] and an association with NEC and genetic disturbances in Paneth cell biology [29]. We mimic this Paneth cell disruption in wild type mice with dithizone treatment, or in genetically susceptible mice by exposure to diphtheria toxin [21–24,30,31]. Dithizone is a heavy metal chelator that disrupts zinc-rich Paneth cells by leading to an upregulation in autophagy pathways [31]. In contrast, our *PC-DTR* strain of mice has a human diphtheria toxin receptor bound to the Paneth cell-specific cryptdin 2 promoter [30–32], and exposure of these mice to diphtheria toxin induces Paneth cell-specific apoptosis. Following either method of Paneth cell disruption, enteral exposure of *Klebsiella pneumoniae* at a concentration of 1 × 1011 colony forming units (CFU)/kilogram body weight to induce bacterial dysbiosis leads to consistent and rapid intestinal pathology that mimics human NEC [26]. Although this model represents two risk factors for NEC (intestinal immaturity and microbial dysbiosis) injury is induced while the mice are not fed.

Infants fed formula are at significantly more risk for NEC than those fed human milk [17,33–35]; however, the mechanism behind this discrepancy is unknown. Human breast milk contains many unique components including nutrients, immunoglobulins, growth factors, hormones, sugars, and proteins that nurture and help to immunologically protect the developing infant. In this fashion, human breast milk is tailored to feed term infants and has everything needed for these infants to grow and succeed [36,37]. However, the caloric requirements necessary to maintain the fetal growth trajectory after birth in preterm infants cannot be achieved by breast milk alone and thus requires fortification from an external source [38,39]. Providing adequate nutrition to preterm infants is critical as there is considerable evidence to support a relationship between poor post-natal growth and later development of neuro-developmental sequelae [40–42].

One side effect of breast milk fortification is a significant increase in osmolality [43]. Osmolality is a measure of the osmoles of solute per kilogram of solvent, with osmoles being defined as a unit of osmotic pressure equivalent to the amount of solute that dissociates in solution to form one mole of particle [44]. The osmolality of infant formula is important as it has been suggested that ingestion of hyperosmolar feeds may lead to NEC [45–47]. While this suggestion was first proposed almost 50 years ago, the understanding of the effect that osmolality has on the immature intestine remains limited. Two recent studies have shown that formula feeding induces changes in intestinal microcirculation and that the associated injury is not osmolality-dependent [48,49]. However, these studies used five- to nine-day-old mice which have not yet developed Paneth cells [20,50] and thus could represent a more immature intestinal epithelium than what is seen in infants who are most susceptible to developing NEC [2,51,52]. To help clarify these gaps in knowledge, we set out to examine the effect of adding formula feeding to our NEC model and to understand if formula induced injury was osmolality dependent. Our novel data show that the addition of formula feeding to either dithizone- or diphtheria toxin-induced Paneth cell disruption eliminated the need to induce dysbiosis to cause NEC-like injury. We additionally show that intestinal injury and markers of inflammation were osmolality-dependent, but not necessarily microbiome dependent. Furthermore, the effects observed were dependent on the method of increasing osmolality. These data expand our understanding of how formula feeding may increase the risk of developing NEC in the preterm infant.

#### **2. Methods**

#### *2.1. Animals and Feeding Protocols*

C57BL/6 mice were bred at the University of Iowa under standard conditions according to protocols approved by the Institutional Animal Care and Use Committee (Protocol #8041401) which was approved on 06/11/2018. Original founders were purchased from Jackson Laboratories (Bar Harbor, ME, USA). All mice were dam fed prior to experiments and, unless otherwise indicated, experiments were conducted with P14–P16 mice. All animals were dam fed prior to experimentation. On the day of experimentation, animals were separated from their mothers and maintained in a temperature and humidity-controlled chamber for the duration of the experiment. Formula feeds [23,53] were prepared and homogenized as previously described from their elemental components. Osmolality was measured using an OsmoPro Multi-Sample Micro-Osmometer (Advanced Instruments, Norwood, MA, USA). High osmolality formulas were achieved by adding Mannitol (MAN; Sigma-Aldrich, St. Louis, MO, USA), or by tripling the salt concentration of rodent milk substitution formula (RMS). Sham animals were given equal volumes of saline (Sigma-Aldrich, St. Louis, MO, USA). All prepared formulas and feeding solutions were given via a 24 × 1 W/1-1/4 blunt animal feeding needle (Cadence, Inc.) and a BD one ml Tuberculin syringe (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Mice were gavage fed 250 μL every three hours × four feeds with one of the following formulations: saline alone; RMS, MAN in saline, NaCl in saline (SALT), RMSMAN added, or RMSSALT (Table 1). Mice were weighed prior to the first feed and following the last feed. Mice were monitored for 3 hours after the final feed and were then euthanized for tissue harvesting. All experiments were performed on at least two separate occasions using at least two different litters. All controls were littermates from experimental animals.

**Table 1.** Definitions of the abbreviations for the feeds used in this paper along with the associated osmolality of each feed type.

#### *2.2. Formula NEC Models*

NEC was induced in postnatal day P14–P16 C57Bl/6J mice by giving an intraperitoneal injection of 75 mg/kg body weight dithizone (Sigma) dissolved in 25 mM LiCO3, or an equivalent volume of LiCO3 buffer alone. Depending on the group being considered, 250 μL of RMS (regular formula NEC), RMSSALT (high osmolality formula NEC), or saline (control animals) was fed through oral gavage one hour prior to injecting with dithizone and every three hours for a total of four feeds. Mice were sacrificed and tissues were harvested thirteen hours after the first RMS/saline gavage. NEC was induced in P14–P16 *PC-DTR* mice by giving an intraperitoneal injection of diphtheria toxin at a concentration of 40 ng/g body weight and oral gavage of saline (control animals) or RMS (formula NEC) one hour prior to injection as well as every three hours for a total of four feeds. Mice were sacrificed at sixteen hours after the first oral gavage and tissues were harvested. Similar to above, all experiments were performed on at least two separate occasions using at least two different litters and all controls were littermates from experimental animals.

#### *2.3. Serum and Cytokine Analysis*

Blood was obtained from the facial vein at the time of euthanasia and serum was processed as previously described [24,54]. Serum samples were used for cytokine (KC-GRO (mouse ortholog to IL-8), TNF-α, IL-6, IL-1β, and IL-10) quantification using a Meso-Scale Discovery V-Plex assay (Meso-Scale, Gaithersburg, MD) on a Mesoscale Sector Imager 2400. Serum osmolality was quantified using an OsmoPro Multi-Sample Micro-Osmometer (Advanced Instruments, Norwood, MA, USA).

#### *2.4. Microbiota Analysis*

Mice were sacrificed according to institutional guidelines at the University of Iowa and ceca were harvested for DNA extraction using the Zymo Fecal/Soil Microbe MiniPrep kit (Zymo Research, Irvine, CA, USA) as previously described [55,56]. Amplification and sequencing were performed on the 16s rRNA V4 domain using a barcoded forward primer 515F (5 -NNNNNNNNGTGTGCCAFCMGCCGCCGCGGTAA-3 ) and the reverse primer R806 (5 -GGACTACHVGGGTWTCTAAT-3 ). The master mix for amplification of the DNA was made with 1x GoTaq Green Master Mix (Promega, Madison, WI, USA); 1 mM MgCl2; 200 nM 806R reverse primer; 200 nM 515F forward primer with barcode; nuclease free H20; and DNA. The thermocycler parameters for the PCR reaction were as follows: 94 ◦C for three minutes; 25 cycles of 94 ◦C for 45 s, 50 ◦C for 60 s, and 72 ◦C for 90 s, followed by 72 ◦C for 10 min and a 4 ◦C hold period indefinitely. PCR amplicons were run via gel electrophoresis to confirm amplification and then were pooled to relatively equal amplification intensities. The pooled amplicons were then resuspended in 2X the volume of Binding Buffer NTI from the Nucleospin PCR and Gel Purification Kit (Macherey-Nagel, Düren, Germany). The Nucleospin PCR Purification protocol was followed according to the manufacturer instructions (Macherey-Nagel, Düren, Germany). The purified pools were then quantitated using QuantIT dsDNA High Sensitivity Assay (Invitrogen, Carlsbad, CA, USA). Libraries were then submitted to the UC Davis DNA Technologies Center for 2 × 300 paired end Illumina MiSeq sequencing. Sequence data was analyzed using QIIME 1 (University of Colorado, Boulder, CO, USA, version 1.9.1) [57]. Sequences were first quality filtered and demultiplexed. Then, UCLUST (drive5.com, Tiburon, CA, USA) was used to assign operational taxonomic units (OTUs) based on a 97% pairwise identity. The OTUs went through a secondary filtration for low abundance OTUs at a 0.005% threshold. After both filtrations, the Ribosomal Database Project database (Michigan State University, East Lansing, MI, USA) was used to taxonomically classify the OTUs and compare with a representative set of the Greengenes 16s rRNA database (Second Genome, South San Francisco, CA, USA, gg\_13\_5 release). PyNAST (University of Colorado, Boulder, CO, USA) was used to align the OTU sequences and construct a phylogenetic tree for further β diversity analysis. Unweighted and abundance weighted UniFrac distance was used to calculate the β diversity. Samples were clustered based according to distances between samples.

#### *2.5. Injury Scoring*

Ileal samples were stained with hematoxylin and eosin to determine injury scores. For NEC models, injury was scored based on a standardized scale as we have described previously [22–24,31] from zero (healthy intestine) to four (full thickness necrosis). Scores of two or greater (separation from the basement membrane and disruption of the villus core) were considered significant for NEC. General non-NEC intestinal injury was assessed by two separate blinded investigators on a three-point scale evaluating villus integrity and separation from the basement membrane as previously described [55,58]. A score of zero was used to describe normal mucosa. Mild injury (score of one) encompassed the development of subepithelial Gruenhagen's space, vacuolization or subepithelial lifting limited to the lamina propria or tips of villi. Severe injury (score of two) involved epithelial lifting and vacuolization greater than half of the villi, villi distortion, or mucosal ulceration and disintegration of the lamina propria.

#### *2.6. Statistical Analysis*

All experiments were performed in at least triplicate and all experiments used animals from at least 2 separate litters. Specific sample sizes are denoted in the Figure legends. ANOVA and non-parametric Kruskal-Wallis testing was performed to determine statistical significance using Graph Pad Prism v8. Significance was set as *p* < 0.05 for all experiments.

#### **3. Results**

#### *3.1. Formula Feeding in Combination with Dithizone Treatment Results in Inflammation and Removes the Requirement for Bacterial Exposure to Induce NEC-Like Injury in the Immature Murine Intestine*

Our previous studies have shown that induction of Paneth cell dysfunction followed by exposure to an enteral bacterial challenge induces NEC-like injury [22,24,54]; however, as opposed to several other common models of NEC [13,59,60], our model does not include formula feeding as a required component. To understand the effect of adding formula exposure to the Paneth cell-disruption model of NEC, we fed a separate group of mice 250 μL of prepared rodent milk substitute (RMS) [23,53,56] every three hours for four feeds prior to and during injury induction and compared them to controls. NEC-like injury was scored as previously described [21–24,31]. Interestingly, animals fed RMS no longer needed additional bacterial exposure to induce significant levels of intestinal injury (Figure 1A). Rather, exposure to enteral RMS feeds, in addition to induction of Paneth cell disruption alone, was enough to induce NEC-like injury equivalent to mice that were exposed to both Paneth cell disruption and *Klebsiella*-induced dysbiosis. Representative histology of all treatments can be seen in Figure 1B. To determine if RMS feeds were inducing inflammation, serum samples were collected at the end of the experiment and measured for Infγ, IL-10, IL-1β, IL-6, KC-GRO (murine homologue of IL-8), and TNF, which are cytokines often associated with intestinal injury and human NEC [61]. Exposure to RMS significantly increased serum levels of all cytokines (*n* > 7 for all groups, *p* < 0.001 for all cytokines measured) (Figure 1C).

**Figure 1.** Formula feeding induces inflammation and removes the requirement for bacterial exposure to induce necrotizing enterocolitis (NEC)-like injury. C57Bl/6J mice (14–16 days old) were given an

intraperitoneal injection of dithizone (75 mg/kg), a gavage feeding of Klebsiella pneumonia, the combination of both, or sham controls (*n* > 7 for all groups). Only the group given both treatments had significantly elevated injury scores. A second group of mice was given the same treatments but was also fed four times with 250 μL/feed of prepared rodent milk substitute (RMS). In this group, both the dithizone alone and the dithizone plus Klebsiella groups had significant injury, showing that adding RMS feeds eliminated the requirement for bacterial exposure (*n* > 7 for all groups, p values as shown) (**A**). Representative histology is shown (**B**). Serum samples were obtained from sham and sham-RMS mice and quantified for IL-10, IL-6, KC/GRO, and TNF. Sham-RMS mice had significantly higher serum levels of all cytokines than non-RMS control groups (*p* < 0.001 for all cytokines, *n* > 7 for all groups) (**C**).

#### *3.2. Paneth Cell Disruption*/*Formula-Induced NEC is not Dithizone-Dependent*

We next sought to determine if the method of Paneth cell disruption in combination with RMS impacted the level of injury and inflammation. P14–P16 *PC-DTR* mice were intraperitoneally treated with 25 ng/g body weight diphtheria toxin to induce Paneth cell apoptosis in addition to four 250 μL RMS feeds given via gavage every three hours for a total of four feeds. Similar to the dithizone/RMS model, diphtheria toxin/RMS treatment induced significant increases in intestinal injury without needing the addition of *Klebsiella*-induced dysbiosis (*n* > 5 for all groups, *p* < 0.0001) (Figure 2A). Representative histology of all treatments can be seen in Figure 2B. We additionally quantified the inflammatory cytokines present in the serum of the *PC-DTR* mice and found significant increases in IL-6 and KC-GRO (Figure 2C).

**Figure 2.** Formula feeding along with Paneth cell disruption resulting in NEC-like injury in the absence of bacterial dysbiosis is not dependent on the method of Paneth cell disruption. *PC-DTR* mice on a C57Bl/6J background (14–16 days old) were given an intraperitoneal injection of diphtheria toxin (40 ng/g), a gavage feeding of 1 × 108/g Klebsiella pneumonia, the combination of both, or RMS in place of K. pneumoniae (*n* > 5 for all groups). Mice fed formula along with diphtheria toxin-induced Paneth cell disruption experienced significant NEC-like injury in the absence of Klebsiella-induced dysbiosis (**A**). Representative histology is shown (**B**). Serum samples obtained from mice before tissue harvest depict significant increases in IL-6 and KC-GRO compared to controls in both dithizone/RMS and DTX/RMS (*n* >10, *p* < 0.001) (**C**).

#### *3.3. Immature Murine Generalized Intestinal Injury and Newborn Mortality Induced by Formula Feeding Is Osmolality-Dependent*

RMS has an osmolality of 721 mOsm/kg H2O, which is more than twice as high as either Pedialyte (250 mOsm/kg H2O), saline (273 mOsm/kg H2O), or most mammalian milk (300 mOsm/kg H2O) [44,53]. To determine if isolated osmolality was responsible for the changes we observed in our NEC model, we fed mice every three hours for a total of four feeds with one of the following: saline (273 mOsm/kg H2O), ad libitum dam feeding (300 mOsm/kg H2O), RMS (721 mOsm/kg H2O), 10% MAN in saline (873 mOsm/kg H2O), or RMS + 10% MAN (RMSMAN) (1491 mOsm/kg H2O). Importantly, animals fed the highest osmolality formula (RMSMAN) had a 50% mortality (most of them prior to the fourth feed) compared to 100% survival for all other feed types (Figure 3A). As mannitol can act as a significant diuretic, we also monitored weights of the animals at the beginning and end of experimentation (Figure 3B). While dam fed animals experienced a significant increase in weight (5.4% increase, *p* = 0.009), sham animals had negligible weight change. Animals fed RMS alone had a slight but non-significant weight increase (2.1%, *p* = 0.27). Animals fed MAN exhibited a significant loss of weight (−13.6%, *p* < 0.0001), as did animals fed RMSMAN (−7.8%, *p* < 0.0001). To quantify generalized intestinal injury, distal ileal sections were harvested at time of euthanasia and scored by two separate blinded investigators on a three-point scale of small intestinal injury described previously [55,58] that is distinct from the NEC injury scoring system (Figure 3C). Injury scores were significantly higher in the RMS, MAN, and RMSMAN groups compared to sham controls (*p* = 0.0023, 0.0009, and < 0.0001 respectively) (Figure 3D).

**Figure 3.** Exposure to increasing osmolality induces significant intestinal injury and mortality. C57BL/6J mice were given four 250 μL feeds with one of the following: sham Pedialyte (250 mOsm/kg H2O), dam feeds (300 mOsm/kg H2O), RMS (721 mOsm/kg H2O), MAN in saline (873 mOsm/kg H2O) or RMS + 10% mannitol (RMSMAN) (1491 mOsm/kg H2O). Mice fed RMSMAN had a 50% mortality compared to all other groups (**A**). Mouse weight significantly increased in dam fed animals (5.4% increase, *p* = 0.009) compared to controls. Animals fed MAN exhibited a significant loss of weight (−13.6%, *p* < 0.0001), as did animals fed RMSMAN (−7.8%, *p* < 0.0001) (**B**). Intestinal injury was determined on a three point injury scale as shown in (**C**). Intestinal injury scores were significantly higher in the RMS, MAN, and RMSMAN groups compared to sham controls (*p* = 0.0023, 0.0009, and < 0.0001 respectively) (**D**).

Next, we wanted to assess the impact of these different osmolality feeds on serum inflammation (Figure 4A). Serum samples were obtained at the time of euthanasia and quantified for the presence of IL-6, IL-10, TNF, and KC-GRO. Sham and dam feeds showed similar levels of all four cytokines. RMS feeds significantly increased IL-6, TNF, and IL-10 levels compared to sham controls. Exposure to MAN feeds caused significant serum elevations of KC-GRO and IL-10, while feeding with the high osmolality RMSMAN induced significant elevations in all four cytokines (n > 5 in all groups and *p* < 0.05 with specific *p* values shown). Lastly, we examined serum osmolality to ascertain if enteral feeds were impacting the animal systemically (Figure 4B). Serum was drawn from animals at time of euthanasia

and measured for osmolality. Feeds with RMS, MAN, or RMSMAN all induced statistically significant elevations in serum osmolality, although the highest elevations were in feeds containing mannitol (n > 5, *p* < 0.0001 for all comparisons).

**Figure 4.** Exposure to increasing osmolality induces significant increases in serum inflammation. Serum samples were obtained at the time of euthanasia and quantified for the presence of IL-6, KC-GRO, TNF, and IL-10 (A). No significant differences were seen between sham and dam feeds. RMS feeds significantly induced IL-6, IL-10, and TNF levels. MAN feeds significantly increased serum levels of KC-GRO and IL-10, while RMSMAN feeds significantly increased serum levels of all cytokines evaluated (*n* > 5 for all groups, p values as shown) (**A**). Serum osmolality was also quantified to ascertain if enteral feeds were impacting the animal systemically. Feeds with MAN, RMS, or RMSMAN all significantly increased serum osmolality, although the highest elevations were in feeds containing mannitol (*n* > 5, *p* < 0.0001 for all comparisons) (**B**).

#### *3.4. Exposure to Mannitol-Increased Osmolality Induced Significant Alterations in the Composition of the Cecal Microbiome in the Immature Intestine*

One possible mechanism responsible for the osmolality-induced injury and inflammation was through alteration of the intestinal microbiome. To analyze this, cecal samples were collected following euthanasia and quantified for microbial composition. All mice receiving feeds experienced shifts in their cecal bacterial flora compared to those receiving only saline (Figure 5A). Mice fed RMS alone had a significant decrease in Firmicutes species similar to what was seen in dam fed mice. Interestingly, there were no differences in the microbiome between mice fed formula and mice that were dam fed. Mice fed either MAN or RMSMAN had significant increases in Proteobacteria with compensatory decreases in the relative number of Bacteroidetes and Firmicutes species. Principle coordinate analysis revealed that animals fed diets containing mannitol (MAN, RMSMAN) had microbiome compositions that were discrete from those fed diets not containing mannitol (Sham, Dam, RMS) as depicted by the distinct clustering of the samples (Figure 5B). Within the Proteobacteria phylum, the families with the biggest increase in the MAN condition compared to sham or dam fed was Enterobacteriaceae and Pasteurellaceae (Figure 5C). Interestingly, in the RMSMAN condition trending increases were seen compared to sham or dam fed to approximately the same amount in Alcaligenaceae, Helicobacteraceae, Enterobacteriaceae, Pasteurellaceae, and Pseudomonadaceae (Figure 5C).

**Figure 5.** Exposure to increased osmolality induced significant alterations in the composition of the cecal microbiome in the immature intestine. Feed substance induced alterations in the microbial composition of the cecum. Dam fed and RMS fed mice had significantly less Firmicutes than sham controls, while mice fed MAN and RMSMAN had a shift in the microbiome resulting in a significant increase in Proteobacteria composition (*n* = 9 animals per group, \* represents a significant change from sham controls where *p* < 0.025) (**A**). Principal coordinate analysis revealed that animals fed mannitol had distinct clustering of their flora compared to all other groups without mannitol in their feeds (**B**). Within the Proteobacteria phylum, the MAN condition resulted in increases in Enterobacteriaceae and Pasteurellaceae compared to controls (**C**). In the RMSMAN condition, increases were seen, compared to controls, to approximately the same amount in Alcaligenaceae, Helicobacteraceae, Enterobacteriaceae, Pasteurellaceae, and Pseudomonadaceae (**C**).

#### *3.5. Osmolality-Induced E*ff*ects Are Dependent on the Methodology Used to Increase the Solute Level*

To determine if increases in injury, inflammation, and bacterial dysbiosis were dependent on MAN or high osmolality, we created a formulation of RMS using three times the salt concentration in place of mannitol to induce a second method for obtaining a high osmolality (RMSSALT) (982 mOsm/kg H2O) feed. Experiments were performed as above using triple salt (SALT) in place of MAN.

As opposed to feeding with MAN, SALT feeds induced no mortality (data not shown as all animals survived), nor significant weight loss (Figure 6A). Further, no change in serum osmolality was seen from controls (Figure 6B). In looking at effects of SALT feeds on serum inflammation, feeding with SALT alone (equivalent salt concentration dissolved in saline) (581 mOsm/kg H2O) had no significant effects on TNF, KC-GRO, IL-10, or IL-6 compared to controls, while feeding with RMSSALT (982 mOsm/kg H2O) significantly increased serum TNF, KC-GRO, and IL-6 levels (Figure 6C). Importantly, while RMSSALT feeds induced cytokine elevation, they were markedly less than what was seen from RMSMAN feeds (Figure 6C—data combined from 4A and 6C for ease of comparison). In quantifying intestinal injury, feeds with both SALT and RMSSALT had significantly increased injury scores compared to sham controls and were similar to those seen with RMS alone (Figure 7A). As was seen in our MAN experiments, the feeds with the highest osmolality (RMSSALT) had the greatest percentage of maximal injury scores, however, as was seen in our cytokine levels, feeds with RMSSALT did not reach the same degree of injury as that seen in RMSMAN. Interestingly, the microbiome showed no significant differences in any phyla when treatment groups were compared to the dam or to sham fed mice (Figure 7B). Although we did not see any shifts at the phylum level, when looking at changes in Proteobacteria families we did see trending increases in both Enterobacteriaceae and Pasteurellaceae in the SALT and RMSSALT conditions compared to sham- and dam fed mice (Figure 7C).

**Figure 6.** Osmolality-induced effects are methodology-dependent. To determine if our results were due to osmolality or to MAN, an additional high osmolality feed was generated by adding three times the salt concentration to standard RMS (RMSSALT). Feeds with SALT and RMSSALT did not show the significant weight loss seen with MAN feeds (**A**), and no change in serum osmolality was observed in SALT or RMSSALT fed groups when compared to controls (**B**). SALT feeds had no significant impact on serum cytokine levels, while RMSSALT feeds induced significant increases in IL-6, KC-GRO, and TNF compared to sham controls (*n* > 4 for all experiments, *p* = 0.0019, 0.0092, and 0.0003 respectively) (**C**). The far-right panel shows the combined MAN and SALT data for more direct comparison of the degree of inflammation generated by each feed type.

**Figure 7.** Alteration of the microbial composition is methodology-dependent, but intestinal injury is methodology-independent and is related instead to the height of osmolality achieved. Similar to our high osmolality MAN groups, feeding with either SALT alone (581 mOsm/kg H2O) or RMSSALT (982 mOsm/kg H2O) had significantly increased generalized injury scores compared to controls (*n* = 7, *p* = 0.0196 and < 0.0001 respectively) (**A**). However, no significant changes in microbial phylum composition were seen when comparing SALT and RMSSALT feeds to controls (**B**). When comparing changes in the Proteobacteria families similar to the comparison made in the mannitol conditions, trending increases were seen in both Enterobacteriaceae and Pasteurellaceae in the SALT and RMSSALT conditions compared to sham- and dam fed mice (**C**).

#### *3.6. High Osmolality RMS Deceased Survival When Included in Dithizone Paneth Cell Disruption and Formula Feeding NEC Model*

Our data shows that formula feeding along with Paneth cell disruption results in significant increases in inflammatory cytokines and in NEC-like injury. In addition, our data shows that exposure of the immature intestine to high osmolality formula (without Paneth cell disruption) significantly increases generalized intestinal injury and serum inflammation when compared to feeds with lower osmolality. Based on this data, we wanted to study the impact of high osmolality formula in one of our newly described RMS NEC models to determine the impact on survival. We disrupted Paneth cells using dithizone and gavage fed 250 μL of RMS with or without high salt to increase the osmolality (as seen in Figures 1 and 2). We found that in these conditions, mice that were fed high osmolality formula had higher mortality rates (93% mortality) compared to their regular RMS fed counterparts (75% mortality) (Figure 8).

**Figure 8.** High osmolality formula combined with Paneth cell disruption results in increased mortality compared to regular formula. C57Bl/6J were injected with dithizone or an equivalent volume of LiCO3 (controls animals) and exposed to feeds with Pedialyte, 1 × 1011/kg *Klebsiella pneumonia*, regular RMS, or RMSSALT (*n* = 11 Sham, 36 Dith+Kleb, 28 Dith+RMS, 28 Dith+RMSSALT). Animals exposed to RMSSALT had increased mortality (93%) compared to those fed regular RMS (75%) or gavage of *K. pneumonia* (14%).

#### **4. Discussion**

The goal of this study was to determine the impact of formula feeding in combination with Paneth cell disruption and dysbiosis on rates and severity of injury to the immature intestine. Our secondary goals were to determine if high osmolality feeds would impact the immature intestine's susceptibility to injury, inflammation, or alterations in the microbiome composition, and to determine if high osmolality would exacerbate mortality in a formula NEC model. Our desire was to address gaps in knowledge regarding the mechanisms behind the increased rates of NEC in infants fed formula as well as questions regarding inconsistencies in data about the role of high osmolality and subsequent susceptibility to develop NEC. Our initial hypothesis was that formula feeding would exacerbate NEC-like injury in the Paneth cell-disruption NEC model. Interestingly, we found that formula feeding along with dithizone or diphtheria toxin to induce Paneth cell disruption caused NEC-like injury in our mice while eliminating the requirement of inducing a secondary bacterial dysbiosis. We further hypothesized that increased osmolality would lead to increased intestinal injury and inflammation through an alteration of the intestinal microbiome. Our novel data show that mice fed high osmolar feeds experience increased serum inflammation and increased levels of intestinal injury compared to sham controls. However, we found that these effects were independent from alterations in the microbiome. Finally, we hypothesized that high osmolar formula would exacerbate mortality seen in our formula NEC model and this was supported by our data. These data are critical to NEC biology and the field of neonatology as the imperative task of providing adequate nutrition to preterm infants requires the fortification of feeds to achieve the caloric requirements to maintain the fetal growth trajectory. Our data adds important insight into the role that formula feeds, and specifically high osmolar formula feeds, have on susceptibility to injury of the immature intestine.

The field of neonatology has advanced tremendously over the past few decades, yet despite these advances there has been slow progress on elucidating the pathophysiology behind NEC. While human breast milk is protective against NEC [17,35], it remains unknown why. The suggestion that hyperosmolality of prepared formulas could induce NEC was first postulated in the 1970s. In 1975, Santulli et al. published a case series reviewing 64 infants with NEC born from 1955 through June 1974. As part of the conclusion of this seminal work, Santulli suggested that hyperosmolar feeds may be causative of NEC as some of the infants who developed NEC had received hyperosmolar feedings (750 mOsm/L) [44,46]. That same year, Book et al. published a prospective study of 16 infants weighing less than 1200 g who were randomized to receive either elemental formula or cow milk formula [45]. Seven of eight (87.5%) infants fed the elemental formula (650 mOsm/L) and two of eight (25%) fed the standard cow milk formula (359 mOsm/L) developed necrotizing enterocolitis (*p* < 0.02). Based on these data, the authors concluded that the hypertonicity of the elemental diet may have contributed to the increased incidence of NEC in infants fed this formula. Following these two manuscripts, the American Academy of Pediatrics (AAP) in 1976 developed a recommendation that the osmolarity of infant formula should be less than 400 mOsm/L [47]. As osmolarity is defined as the number of solute particles in 1L of solvent and osmolality is the number of solute particles in 1kg of solvent, the AAP recommendations approximates to an osmolality of 450 mOsm/kg H2O [62]. This was a consensus view based on the observation that the milk of most mammalian species has an osmolarity of approximately 300 mOsm/L and that hyperosmolar formulas may be a causative factor in NEC. Despite (or perhaps because of) this consensus statement, little data has been generated to support the theory that osmolality is detrimental. Recent works by the Pierro lab have begun to address this gap in understanding. Their work utilizes five- to nine-day-old mice who were given systemic hypoxia over several days as well as hyperosmolar feeds laced with LPS three times a day. In their studies, the Pierro lab has shown that formula feeding induces changes in intestinal microcirculation and that the associated injury is not osmolality-dependent [48,49]. However, our data differs in that our mice were significantly older from a developmental pattern standpoint [20] and our studies used a much broader range of osmolalities as well as two different methods of altering feed osmolality. In this way, our data expands and adds to the important previous work of the Pierro group.

Although current neonatal practice is to use low osmolality feeds in the neonatal intensive care unit (NICU), these formulas are frequently fortified with supplemental protein, as well as other substances essential for growth such as vitamins or minerals to provide adequate nutrition. It is important to realize that use of the fortifiers and other supplements greatly increase osmolality. Our data show that feeding just four feeds of high osmolar formula to neonatal mice can provoke significant increases in serum inflammation and in epithelial damage in the immature small intestine. This appears to be dose-dependent, as feedings with higher osmolality had higher inflammation and injury scores. While breast milk, amniotic fluid, and Pedialyte all have osmolalities below 350 mOsm/kg H2O, our formulas were significantly higher: SALT (581 mOsm/kg H2O); RMS (721 mOsm/kg H2O); MAN (873 mOsm/kg H2O); RMSSALT (982 mOsm/kg H2O); and RMSMAN (1491 mOsm/kg H2O). Importantly, our injury scores for RMSSALT and MAN alone (982 and 873 mOsm/kg H2O, respectively) were similar, as were the injury scores for RMS alone and SALT (721 and 581 mOsm/kg H2O, respectively). The highest generalized injury scores were observed with the highest osmolality formula, RMSMAN (1491 mOsm/kg H2O). While the osmolality of acceptable NICU feeds is below the levels that induced injury in our model, the formula and standard fortifiers commonly used do not take into consideration other substances that are often used with feeds in the NICU. A recent paper by Chandran et al. examined fourteen common medications utilized in the NICU [63]. Of these medications, nine had osmolalities too high to measure and the five additional medications were all above 450 mOsm/kg H2O. This list included iron, multi-vitamins, NaCl, NaPO4, and KPO4, which are all common additives to feeds. To keep the osmolality below 450 mOsm/kg H2O, the medications required significant dilutions of up to 1:40 when mixing the medication in preterm formulas and 1:250 when mixing with fortified expressed breast milk [63].

We observed a trend of increased injury being associated with higher osmolality feeds, but interestingly this trend was independent of the method for generating higher osmolality. While we were concerned that MAN would cause injury through dehydration, use of high salt formula did not impact weight while still inducing intestinal damage. This contrasts the work by Miyake et al. [48]. In this work, there were no differences seen in injury generation between formulas with an osmolality of 849 and 325 mOsm/kg H2O. However, all animals also underwent exposure to LPS four mg/kg/day and hypoxia three times a day for 10-min time periods to induce NEC-like injury; thus, it is possible that the injury sustained in the hypoxia-formula model of NEC is severe enough that it would overwhelm any injury patterns seen in high osmolality alone. Although there is still conflicting data on the effect

of formula osmolality and its relation to NEC likely due to conditional differences, our data highlights the danger of high osmolality not just in causing intestinal injury and inflammation, but also mortality as well when high osmolality formula is combined with Paneth cell disruption. Interestingly, our data suggested that the injury, inflammation, and mortality observed was related to the osmolality itself and could occur without changes in the microbiome, suggesting, at least for the microbiome, what is used to increase the osmolality is more indicative of community type than the osmolality itself. Additionally, it suggests that intestinal injury and inflammatory cytokine levels are likely independent of the microbiome in this particular model as intestinal injury and inflammation can be increased without shifts in the microbial community.

These findings were in direct contrast to our original hypothesis as we predicted increased osmolality would lead to increased intestinal injury and inflammation through an alteration of the intestinal microbiome. We were not surprised to find that different feed formulations had differential effects on the microbiome, but we were surprised to find that they were independent of injury scores. One finding was that feeds containing mannitol had significant expansions of the Proteobacteria phyla and more specifically Enterobacteriaceae. This is interesting as many studies have associated similar blooms with development of NEC [10,64]. Furthermore, many studies have linked formula feeding with NEC, and one connection made is that formula feeding tends to result in differential colonization of the infant gut compared to breast feeding. While breast fed infants tend to have a predominance of Firmicutes and Actinobacteria, formula-fed infants tend to have a predominance of Proteobacteria and more specifically Enterobacteriaceae, which can put them at higher risk for bacterial dysbiosis, which is another risk factor for NEC [65]. Another interesting finding was that in salt conditions we did not observe significant changes in the microbiome at the phylum level, but when looking at family level changes, we saw a trending increase in Enterobacteriaceae in the higher osmolality SALT and RMSSALT conditions. These conditions were associated with higher generalized injury scores; however, injury scores and inflammation were more consistent on overall osmolality than alterations in the cecal microbiome. One possibility for the increase in specific groups of organisms with mannitol feeds is that many Proteobacteria and Firmicutes species can utilize mannitol as an energy source allowing for a selective expansion of the bacteria that can utilize mannitol, which was supported by our data [66–68]. Additionally, Proteobacteria and Firmicutes species tend to be more tolerant to high salt conditions compared to other phyla of bacteria, which may explain why there were trending increases in some Proteobacteria families [69,70]. These data suggest that although changes in the microbiome were present in both the mannitol and salt related conditions, they likely occurred independent of the inflammation and injury we observed and were not causative in these experiments.

Although we did not observe microbiome alterations between dam fed and formula-fed mice, we did find significant increases in inflammation in formula-fed animals compared to dam fed animals. Formula feeding has been linked with increases in inflammation, which may explain why it can replace bacterial dysbiosis in our model and serve as a secondary hit in other NEC models [18]. Similar to other labs that have shown that formula feeding along with some other secondary form of insult such as hypoxia/hypothermia, LPS, or bacterial insult can induce NEC, we saw that when we induced Paneth cell disruption using either dithizone or diphtheria toxin and followed this with formula feeding of RMS we saw significant increases in NEC-like injury [13,15,18,48,59]. Unsurprisingly, the injury we observed was slightly different based on the type of Paneth cell disruption induced. Slightly higher NEC scores were found in the dithizone RMS NEC model and the damage in the intestine was mainly confined to the base and the core of the villi. While the scores were still significant in the diphtheria toxin RMS NEC mice, the scores trended to be slightly lower on average compared to the dithizone RMS NEC. Additionally, the damage pattern in the intestine included both the base of the villus as well as vacuolized cells at the top of the villus down compared to the damage pattern in the dithizone RMS NEC. This supports the findings of Lueschow et al. in 2018 showing that the NEC scores with diphtheria toxin and bacterial gavage, although still significant, trended slightly lower on average compared to the scores seen with dithizone and bacterial gavage [31]. Additionally, the difference

in damage patterns of the intestine between dithizone and diphtheria toxin that we observed while formula feeding is complemented by the two different types of Paneth cell disruption described in Lueschow et al., 2018 where dithizone induces upregulation of autophagy pathways while diphtheria toxin induces apoptosis [31,71]. The findings that formula can replace induction of bacterial dysbiosis in a Paneth cell disruption NEC model highlights the potential dangers of formula feeding in premature infants at heightened risk for NEC.

Ultimately, the results of this paper demonstrate that formula feeding can induce NEC-like injury when combined with Paneth cell disruption without induction of bacterial dysbiosis, which is likely due to the inflammatory nature of the RMS formula without any additives. Additionally, high osmolality formula without Paneth cell disruption results in increased generalized intestinal injury and inflammation to a greater degree than formula feeding on its own in a microbiome independent manner. When high osmolality formula was combined with Paneth cell disruption, higher mortality rates were seen compared to when RMS alone was combined with Paneth cell disruption. Furthermore, the results show that changes in the microbiome can occur independent of injury and inflammation and were not causative in this case, as injury and inflammation were more closely connected with osmolality than changes in the microbiome. This is a critical point to consider as osmolality, microbiome composition, and intestinal injury can be interrelated or completely independent of each other depending on the conditions present. Therefore, as we consider optimized feeds that maximize nutrition for preterm infants, we must be vigilant of what we are giving to make sure we protect their immature intestinal tract.

**Author Contributions:** Conceptualization, S.L.K., J.L.S., S.J.C., S.J.M.; methodology, S.L.R., S.L.K., H.G., J.L.G., S.J.M., validation and formal analysis: S.R.L., S.L.K., H.G., J.L.G., S.J.M.; investigation: S.L.R., S.L.K., H.G., J.L.G., S.J.M.; resources, S.R.C., J.L.G., S.J.M.; data curation: S.R.L., S.L.K., H.G.; writing—original draft: S.L.K., J.L.S., S.J.M.; writing—editing: S.R.L., S.L.K., H.G., J.L.G., J.L.S., S.R.C., S.J.M.; supervision: S.J.M.; funding acquisition: J.L.G., S.J.M.; All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** Support for this work was provided from the NIH, grants HL134850 and HL084207, The American Heart Association, grant 18EIA33890055, and the Stead Family Department of Pediatrics at the University of Iowa, and the Children's Miracle Network.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).


**2.2 Geographical distribution**

Globally, HLB has been considered one of the significant threats to citrus commercial and sustainable production. HLB was confirmed in citrus-producing regions of various countries, such as Nepal, Bangladesh, Thailand, Pakistan, Japan, Vietnam, Cambodia, Laos, Malaysia, Central African Republic, Comoros, Ethiopia, Kong Hong, Kenya, Madagascar, Malawi, Mauritius, Saudi Arabia, Reunion, Rwanda, Yemen, Zimbabwe, Somalia, Tanzania, Swaziland, and various region of United States of America including California, Florida [7, 27]. HLB has been reported in 24 countries and territories in East, South, Southwest Asia, East, and South Africa. Since then, it has been widely spread in other Asian, American, and African countries [27].


*Surface Modification of Titanium Orthodontic Implants DOI: http://dx.doi.org/10.5772/intechopen.100038*

techniques which are effective only initially, indicating that UV photofunctionalization does not merely accelerate the process of osseointegration but also increases the level/ degree of osseointegration [71].

RT values for UV-treated implants were reported to be 50–60% than those of untreated implants [83]. The osseointegration speed index (OSI) calculated as the difference between two ISQ readings at different intervals was reported to be 2–3 times higher for UV-treated implants [86–89]. The biomechanical push-in values assessed for UV-treated implants using a rat model were 2.5–3 times greater than those of untreated implants [63, 64, 71, 90]. In most of the studies the level of osseointegration seen at week 2 around UV-treated implants was equivalent to that seen around the untreated implants at week 8, indicating that UV treatment may have the potential to accelerate the process of osseointegration 4-fold [71]. These results suggest that UV photofunctionalization may be effective in enhancing the anchoring capability of titanium implants.

#### **Figure 12.**

*Peri-implant bone morphogenesis enhanced by photofunctionalization. Low magnification microscopic images of peri-implant tissues around untreated implants (A) and photofunctionalized implants (B). High magnification images of untreated implants (C–E) and photofunctionalized implants (F–H), zooming up the portions in (A) and (B) in each of marginal, cortical, and bone marrow zones. (Taken from: Pyo et al. [83].)*

#### **Figure 13.**

*Scanning electron microscopy images showing energy dispersive x-ray spectroscopy (EDX) mapping (A,B) and EDX spectrum (C,D) of the apical part of the screw at 4 weeks. The Ca/P ratio shows that the mineralized tissue attached to the surface on the screw is bone. Titanium (Ti) was mainly detected in the mapping of the untreated group (A). However, much more bone tissue had attached to the screws in the photofunctionalized group, and mapping showed more Ca and P than Ti, indicating that the surface was more greatly covered by bone tissue than in the untreated group (B). Ca/P ratio (E) and Ca/Ti ratio (F) of the surface of the apical part of the screw at 4 weeks. Both Ca/P ratios were equal and consistent with bone tissue. The Ca/Ti ratio in the photofunctionalized group was extraordinarily greater compared with that of the untreated group, indicating dense and rich bone tissue covering the screw surface (\*\*P < .01). (Taken from: Hirota et al. [72])*

The effects of UV photofunctionalization of implants have been studied in challenging host conditions for osseointegration to simulate clinical situations [64, 65, 66, 69, 91–94]. Ueno et al. reported greater strength of osseointegration in a rat model at both early and late healing stages for UV-treated shorter implants as compared to untreated regular length implants [91]. This suggests that UV photofunctionalization may overcome the loss of anchoring capacity due to reduced length of implants and may allow the use of shorter implants in certain clinical situations. Kim et al. reported enhanced osseointegration in UV-treated implants placed near critical one-wall defects in beagle dogs [85]. Kitajima et al. reported that photofunctionalized implants placed with low, extremely low, or even absent primary stability showed a high success rate eventually

*Surface Modification of Titanium Orthodontic Implants DOI: http://dx.doi.org/10.5772/intechopen.100038*

[92]. Kim et al. and Lee et al., through their studies in rabbit calvarial defects, showed that UV-treatment promoted *de novo* osteogenesis as well as enhanced bone regeneration in critical rabbit calvarial defects [93, 94]. Thus, there is enough evidence to suggest that UV photofunctionalization may play a major role in mitigating challenging/compromised host conditions and aid in enhanced implant integration.

However, of all the studies which have reported the effects of photofunctionalization, only Mehl et al. reported this surface modification technique to be ineffective in enhancement of implant biologic activity [95]. Their *in vivo* study in edentulous minipig jaws revealed that the BSC value for UV-treated implants after 9 months of healing was about 64% only, which is similar to what many studies have reported for conventional UV-untreated implants, suggesting that photofunctionalization had no significant effect in enhancing osseointegration.

#### **3.4 Effects on other implant materials**

A majority of published literature on UV photofunctionalization is based on titanium as the implant material as it is most commonly used. However, there are some studies which have reported the effect of UV treatment on other implant materials as well. *In vitro* analyses of zirconia disks showed that their UV pre-treatment resulted in a physiochemical alteration of surface properties similar to those seen in UV-treated titanium surfaces [96, 97]. Brezavšček et al. showed that osteoconductive capacity of zirconia-based implant materials in a rat model was enhanced by their UV pre-treatment [98]. Shahramian et al. reported that UV treatment of zirconia disks (TiO2-coated and non-coated) promoted platelet activation and thereby hastened blood coagulation [99]. This suggests that UV treatment has the potential to expedite wound healing around plain as well as coated zirconia implants.

Decco et al. reported that UV treatment of sandblasted chromium-cobalt-molybdenum (Cr-Co-Mo) alloy disks resulted in physiochemical alteration of surface properties similar to that of UV-treated titanium [100]. A recent study by Elkhidir et al. on ratderived mesenchymal stem cells (MSCs) showed that UV treatment of gold nanoparticles increased its osteogenic capabilities by enhancing cell functions as well as osteogenic gene expression (Col-1, osteoprotegerin, osteocalcin) and mineralization [101]. All of these studies suggest that photofunctionalization of non-titanium implant materials also enhances their bioactivity and can have varied applications in the future.

#### **3.5 Effects on orthodontic miniscrews**

Despite this technique having been proven effective for all sizes and topographies of titanium implants, its clinical use with orthodontic miniscrews has not yet been investigated thoroughly. An *in vivo* study by Tabuchi et al. in rat femurs evaluated the osseointegration potential of photofunctionalized orthodontic miniscrews [102]. Via biomechanical push-in tests, it was found that displacement of untreated screws was 1.5–1.7 times greater than that of UV-functionalized screws (**Figure 14**). Surface evaluation showed robust bone formation around UV-treated screws with strong elemental peaks of calcium and phosphorus, whereas the tissue around untreated miniscrews appeared thin and showed no clear peak of calcium. In a similar comparative study, the maximum IT and RT values were measured. While the IT values were similar for both groups, the RT values were considerably higher for UV-treated miniscrews. This implied that implant strength at insertion was similar whereas, at removal, the strength of UV-treated miniscrews was much greater. SEM analysis

#### **Figure 14.**

*The anchorage strength of orthodontic miniscrews with and without photofunctionalization. (a) Representative load–displacement curves for untreated and photofunctionalized miniscrews subjected to a lateral tipping load. (b) The amount of miniscrew horizontal displacement under various levels of load; \*P < .05; \*\*P < .01. (Taken from: Tabuchi et al. [102].)*

#### **Figure 15.**

*Scanning electron micrograms of the miniscrews at week 3: (A-J) miniscrews with and without photofunctiolization were compared. (Taken from: Tabuchi et al. [103].)*

revealed that regenerated bone tissue was more intact and contiguous around the UV-treated miniscrews than around the untreated ones, and the miniscrew-bone complex seemed to produce interface failure, and not cohesive fracture (**Figure 15**) [103]. Takahashi et al. studied the stability of UV-functionalized orthodontic miniscrews under immediate loading in growing rats [104]. A significantly less (almost 1/2) screw mobility was observed with the UV-treated miniscrews in both, the unloaded as well as immediately loaded groups. Once again SEM analysis revealed an increased BSC (1.8 times) in the UV-treated miniscrew groups.

Recently, the authors conducted a split-mouth *in vivo* human study for the first time using photofunctionalized miniscrews [105]. They studied the effect of UV

*Surface Modification of Titanium Orthodontic Implants DOI: http://dx.doi.org/10.5772/intechopen.100038*

#### **Figure 16.**

*Representative SEM images of untreated and UV-treated groups from upper, middle and lower regions of miniscrews:* **A***, images taken at 100X magnification,* **B***, images taken at 500X magnification. (Taken from: Rampurawala et al. [105].)*

#### **Table 2.**

*Comparison of Ca/Ti and Ca/P ratios between surfaces of untreated and UV-treated miniscrews in the upper, middle and lower regions.*

photofunctionalization on orthodontic miniscrews using SEM to evaluate the BSC and EDS to evaluate surface element deposition. It was observed that there was increased BSC in lower regions of miniscrews in the photofunctionalized group

(**Figure 16**), but this was not statistically significant. There was also no significant difference between the Ca/Ti and Ca/P ratios of UV-treated and untreated miniscrews (**Table 2**). The results of this study were in agreement with only one previous study that reported a lack of improvement in the biomechanical potential of implants [95].


Preface

Today, information in medical science is increasing rapidly, and with the communication age, accessing information is quite easy. However, with this increasing knowledge, information complexity has emerged. Thus, despite all this increasing knowledge, the interest and need for basic books have not diminished. Blood groups, erythrocyte antigens, and transfusion are fundamental areas of medicine and are related to many disciplines of science like hematology, immunology, surgery, and genetics. This book is a collection of current information related to blood groups and transfusion, and a practical resource for all concerned physicians.

 I would like to thank all the authors and IntechOpen and hope that this book will be a stepping stone for scientists who are rapidly advancing their science journey.

> **Dr. Anil Tombak, MD**  Associate Professor, Department of Internal Medicine-Hematology, Mersin University Medical Faculty, Mersin, Turkey

**1**

Section 1

Clinical Significance of

Blood Groups

## Section 1


3.2.3.  The scenario analysis is conducted with the cost of infrastructure development deducted from agricultural income. The deduction of cost counts as the impact of investment on agriculture or non-agriculture, which does not impact the cost of capital development. This means that agricultural income in the paper, in effect, represents a "net income" that reflects the impact of technological investment costs.

Canal lining aims to improve conveyance efficiency. Conveyance efficiency increases through investment in irrigation infrastructure that reduces canal seepage and increases water delivery to the fields relative to water released from the reservoir upstream. Improvement of conveyance efficiency necessitates irrigation infrastructure and involves deduction from profit. Canal lining cost varies depending on lining material, canal cross-section design, and installation approaches. Based on the Bureau of Reclamation's historical record, the canal lining cost ranges from 14.9 to 37 dollars per square yard [33]. The canal lining cost ranges from up to 2000 dollars per acre-foot of water saved [34]. Multiplying the unit cost of canal lining and surface water divisions yields the total cost. More of surface water withdrawals will be delivered to the field with canal lining, and less water will be "lost" as canal seepage. In the model, as conveyance efficiency increases, the total cost for canal lining will be deducted from agriculture income. The regional canal lining project is a long-term process, so canal lining takes ten years to plan and ten more years to implement. The service life of the canal lining is deemed to be forty years [34]. The canal lining process continues until the total irrigation supply reaches the largest yearly water supply of surface water. The variables involved in the policy test are listed in Table 2.

**Table 2.** Variables setting for IE policy.

Precision irrigation aims to improve field irrigation efficiency. Irrigation systems could be altered to improve field efficiency (e.g., through drip and sprinkler irrigation). High IE irrigation systems typically maintain the same productivity with less water applied or increase productivity with the same water application rate due to more water delivered to the fields being available to crops [35,36]. Field irrigation efficiency refers to irrigation technology and practices that attempt to apply only the water necessary to replenish the amount of water lost through ET and reduce percolation past the root zone. Costs associated with irrigation conservation have been cited for having varied installation costs per acre and operation costs [37]. As summarized in a report of drip irrigation experiments, the cost for design, materials, and installation of a drip irrigation system for the case of a farm in Rincon, New Mexico, which was a 26-acre farm, was 52,000 dollars [38]. The variables involved in the policy test are listed in Table 2.


**6.2 Transcription factor Nrf2**

Transcription factor Nrf2 (Nuclear factor E2–related factor 2) binds to and induces activation of the ARE. Nrf2 was first isolated by an expression cloning procedure using an oligonucleotide containing the NF-E2 DNA binding motif as a probe to screen for closely related proteins (Moi et al., 1994). Nrf2 belongs to the cap-and-collar family of basic region– leucine zipper transcription factors, and is an essential component of the ARE-mediated transcriptional machinery. It has been shown that Nrf2 mediates both the basal and inducible activity of the ARE, and the loss of Nrf2 results in a profound reduction in the enzyme activities of NQO1 and certain GST isoenzymes (Itoh et al., 1997). These observations also correlate well with the ubiquitous expression of Nrf2 at steady-state levels

The Botanical Extract Feverfew PFE Reduces

Fig. 4. Feverfew PFE induces activation of the ARE promoter.

100

0

0

50

100

150

**% Increase in Nrf2 activity**

**over control**

200

250

300

50

100

150

**ARE Promoter**

**% Change over Control**

200

250

300

Fig. 5. Feverfew PFE activates the Nrf2 transcription factor.

100

enhanced the nuclear translocation of Nrf2 by more than 70%.

In order to analyze the effects of Feverfew PFE on the transcription factor, Nrf2, a TransAM ELISA was utilized. Primary human keratinocytes were treated with Feverfew PFE or Sulforaphane for 24 hr, following which the nuclear and cytoplasmic cell lysate fractions were separated. Translocation of Nrf2 was analyzed in the nuclear fractions. Feverfew PFE

172

**Control Feverfew PFE (10ug/mL) Sulforaphane (10uM)**

**Feverfew PFE (10μg/mL) (10μM)**

DNA Damage and Induces DNA Repair Processes 539

164

**Control Feverfew PFE (5ug/mL) Sulforaphane (10uM)**

**Feverfew PFE (5μg/mL) Sulforaphane (10μM)**

233

239

in various tissues and cell lines (Nguyen et al., 2004). As a result, the function of Nrf2 and its downstream target genes are vital for protection against cellular damage induced by oxidative stress or chemicals. Several studies have shown that Nrf2 knockout mice having decreased levels of phase II detoxification enzymes and antioxidant proteins are highly sensitive to cytotoxic electrophiles compared to their wild-type littermates (Lee and Johnson, 2004; Leung et al., 2003). The upregulation of protective detoxification and antioxidant genes by Nrf2/ARE pathway can synergistically increase the efficiency of our cellular defense system.


**4. Discussion**

Early assessment of NAC response to breast cancer and correct differentiation between patients with the complete pathological response (pCR) and without NAC response is the key point in NAC therapy. Proper evaluation is crucial to the future clinical perspectives and therapy for each patient. Obtaining complete pathological response results in better event-free survival (EFS) and overall survival (OS) rates [33,34].

In our study, the sensitivity of low-energy images in forecasting CR reached 33.33%, the specificity was 92.86%, the PPV was 70%, and the NPV was73.58%. After the conversion of subtraction images, the sensitivity of CESM in CR detection in a group of patients after NAC was 85.71%, its specificity was 71.42%, the PPV was 60%, and the NPV was 90.90%. Similar results for subtraction CESM images have been acquired by other authors such as Patel et al. [35] (64 patients, sensitivity: 95%, specificity: 66.7%, PPV: 55.9%, NPV: 96.7%), Iotti et al. [36] (46 patients, sensitivity: 100%, specificity: 84%, PPV: 57%, NPV: 100%), and Barra et al. [37] (33 patients, sensitivity: 76%, specificity: 62.5%, PPV: 86%, NPV: 45.4%). All mentioned studies, including ours, showed that, in the assessment of the CR following NAC, subtraction CESM images reached significantly higher sensitivity. Unfortunately, their specificity was lower. These results demonstrate that imaging techniques, even after intravenous administration of a contrast agent, may not differentiate between residual infiltration lesions and co-existing inflammatory/reactive lesions. A similar problem occurs with MRI, which tends to underestimate residual changes [23].

In their meta-analysis, Tang et al. [38] demonstrated that the pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR) of the pathological response of breast cancer to NAC assessed by CESM were:

0.83 (95% CI, 0.66–0.93), 0.82 (95% CI, 0.68–0.91), 4.66 (95% CI, 2.59–8.41), 0.20 (95% CI, 0.10–0.43), and 22.91 (95% CI, 8.66–60.62), respectively. The same parameters for MRI, which is considered to be the best method for assessing response to NAC, were as follows: sensitivity: 0.77 (95% CI, 0.67–0.84), specificity: 0.82 (95% CI, 0.73–0.89), PLR: 4.35 (95% CI, 3.00–6.33), NLR: 0.28 (95%CI, 0.20–0.39), and DOR: 15.48 (95% CI, 9.97–24.03). Based on these findings, it can be concluded that CESM has the same specificity and higher sensitivity than MRI and is more accurate in the pathological evaluation of NAC response in breast cancer treatment.

The largest pretreatment tumor dimensions in our analysis of low-energy and subtraction CESM images were similar and there was a statistical difference between these modalities (R = 0.89, *p* = 0.01). However, these differences became significant following neoadjuvant chemotherapy (R = 0.55, *p* = 0.01). This was because post-NAC tumors reduce their density and then become difficult to distinguish from glandular tissue based on morphological images alone. On the other hand, the functional information provided by CESM in subtraction images showed that the residual infiltration was visible, and the type of breast tissue did not affect its visualization. Moreover, in our study, the comparison of the measurement of the maximal sizes of residual changes after NAC, evaluated using CESM subtraction images and histopathology, showed a high correlation (R = 0.67, *p* = 0.01). Iotti et al. [36], while comparing the sizes of tumors after NAC with a histopathological examination, proved that CESM showed greater coherence with histopathology than MRI (Lin's coefficient were 0.81 and 0.59, respectively). In contrast, Patel et al. [35] achieved the opposite results, where MRI showed higher compatibility with histopathology than CESM (Lin's concordance coefficient was 0.75 for CESM and 0.76 for MRI; Pearson's correlation was 0.77 for CESM and 0.80 for MRI). The lack of consistency between researchers indicates the need for further research—multicenter, on a larger group of patients, using equipment available on the market from various companies.

In our study, Low-energy images tended to overestimate the dimensions of residual lesions following NAC, while subtraction CESM images tended to underestimate them. Similar results were acquired by Patel et al. [35] and Iotti et al. [36], where CESM and histopathology results underestimated the size of post-NAC tumors by 5mm and 4.1mm, respectively. It should also be emphasized that the underestimation of the dimensions of residual lesions in our study had no impact on the scope of surgical treatment. Since CESM is a method involving vascularization of the tumor foci, the effect of excessive reduction in vascularization around the tumor during NAC may account for the weaker enhancement of the residual tumor mass on follow-up CESM, underestimating the actual dimension of residual lesions. A similar problem arises with MRI, which tends to underestimate residual lesions in follow-up examinations [35,36]. Over-or underestimation of residual disease can also be a result of the fact that, due to neoadjuvant chemotherapy, induction of cellular changes leading to the elimination of cancerous cells occurs before the decrease in tumor size [39,40]. Moreover, after the eradication of cancerous cells in the region of the tumor, the residual mass visible on radiological images may still be present. It consists of fibrotic tissue left after the therapy. To overcome this problem, Xing et al. [41] and Moustafa et al. [42] suggested relying not only on RECIST 1.1 criteria in the evaluation of NAC response but also creating a special mathematical model. It consists of a combination of the measurement of the largest diameter of the target lesion together with subjective identification of the difference in the intensity of the contrast uptake before and after NAC. After that, a combination of the summation of the number of pixels and their intensity within the area of interest before and after NAC is included in this model. It should be noted that, after using this method, CESM remained a method of high sensitivity and specificity in the evaluation of NAC response. Such results prove that CESM is one of the best methods for diagnostic imaging available for the analysis of the response to neoadjuvant chemotherapy.

The histological and molecular heterogeneity of tumors cannot be evaluated with a simple examination with a contrast medium, and biopsies are limited, especially in large tumors. The need to better define the heterogeneity of tumors will involve the aid of new methodologies currently under study, such as radiomics. Radiomics in MRI can be more effective in the diagnosis of breast cancer and in the histological and morphological assessment of tumors. For CESM, a radiomics model achieved a significantly better discriminative ability compared to the standard clinical model (AUC, 0.81 vs. 0.55, *p* < 0.01) [43–45]. In recent years, it has also been suggested to use background parenchymal enhancement (BPE) in the assessment of responses to NAC, which describes the enhancement of the normal breast tissue related to physiological vascularization. La Forgia et al., indicated that BPE is an important aspect that conditions the diagnosis and that it is a potential predictive factor in the response to neoadjuvant cancer therapies in graphic contrast examinations, as already confirmed in MRI and CESM examinations [46–48].

 Subtraction images improve the diagnostic specificity of low-energy images, providing more precise measurement of tumor size as well as the possibility of identifying multifocal diseases, especially in women with dense breast tissue. Thus, the effectiveness of CESM in breast cancer diagnosis is comparable to MRI and is a promising tool to serve as a basic imaging technique in patients with symptomatic breast cancer or for the detection of multifocal and multicentric breast cancers [49–51]. As we have shown in our study, CESM is also a useful and effective method in assessing the pathological response to NAC. Post-NAC treatment monitoring is extremely important for planning surgical treatment, reducing the number of mutilating mastectomies, and replacing them with breast-conserving surgeries. However, the most accurate measurements possible should be made to avoid underestimating the size of the tumor and increasing the extent of the operation. The advantage of CESM over other methods so far available is its precise definition of the tumor before NAC thanks to the directly integrated visualization of suspicious calcifications in the low-energy images and enhancement in the recombined images, which is not possible with MRI [52].

Our study had several limitations. Firstly, this single-institution research was carried out on a relatively small number of female patients due to the limited number of qualifications for NAC. Moreover, all CESM examinations were conducted on a single vendor system. Finally, we did not include patients with HER2-positive cancer in our study due to the lack of public funding for trastuzumab in this center in the years from2016–2019 (no drug prescription government program). There is a need for multi-institutional studies with larger groups of patients free of the limitations described above in the future.


*2.4. Fatty Acid 13C Atomic Enrichment*

Figure 6 shows the atomic enrichment (AE) of the eleven fatty acids over time. Despite the different timing and level of enrichment between the two balloons, the temporal dynamic of fatty acids enrichment remained similar. The 18:2n-6 and 18:1n-9 + 18:3n-3 had the highest AE during the entire experiment. 22:5n-6, 16:0, and finally, 20:5n-3 were next. The less enriched fatty acids in the polar lipid fraction were in decreasing order 22:5n-3, 22:6n-3, 18:4n-3, 18:5n-3, and finally, 18:0 (Figure 6). For the NL (Supplementary Files, Figure S2), 20:5n-3, 22:5n-6, 18:1n-9, 16:0, and 18:2n-6 were always the most enriched. The sequence for the other fatty acids remained close to that of polar lipids. It has to be noted that enrichments of 20:5n-3 and, to a lesser extent, of 22:5n-3 were higher in NL than in PL.

Table 1 explored FA synthesis pathways with regard to their most expected direct precursor. Most ratios were below 1, except for the 20:5n-3/18:5n-3 ratio, which was above 1. Similar patterns were observed in NL (Supplementary Files, Table S5).

**Table 1.** Mean ratio of atomic enrichment (AE) for pairs of FA (FAA vs. FAB) in the polar lipids (PL) (mean ± SD, *n* = 9 sampling dates t8 to t24) for the two enriched balloons (Tl1, Tl2, Tl = *Tisochrysis lutea*).

\* If the AE of the product (B) exceeds the AE of the reactant (A), ratio > 1, then it is necessary to consider another formation process for B. If the ratio is <1, transformation of A into B is considered possible. If the ratio is close to 1, the fatty acids A and B are at the equilibrium in terms of label incorporated, implying B is then synthesized simultaneously or very rapidly from A.

**Figure 6.** Atomic enrichment of 11 main fatty acids in the polar lipid (PL) fraction during a 24 h 13C labelling experiment of the two enriched balloons of *Tisochrysis lutea* (TI1 and TI2, filled circles and filled triangles, respectively).

### *2.5. Identification of Candidate Proteins for PKS Synthesis in T. lutea*

Thirty sequences of potential candidate proteins involved in *T. lutea* PUFA synthesis have been identified and are presented in Supplementary File (Table S6). Only fourteen presented the four main domains potentially coding for the enzymes used in PKS PUFA synthesis pathways: ketoacyl reductase (KR), polyketide synthase (KS), dehydrase/dehydrogenase (DH), and enoyl reductase (ER). Among these sequences, four sequences (TISO\_14962, TISO\_14968, TISO\_14975, and TISO\_14977) were part of the same cluster (group of homologous proteins) and presented multiple KS, KR, ER, and DH domains as well as phosphopantetheine (PP)-binding domains (Figure 7). TISO\_14962 also possessed methyltransferases and thioesterase domains (Figure 7). TISO\_14977 presented a domain acknowledged to be involved in acetyl-CoA synthesis. Within this cluster, TISO\_14973 was also selected, as it contains an atypical domain, specifically recognized as being involved in n-3 PUFA synthesis. Nine other sequences (TISO\_04539, TISO\_06404, TISO\_06537, TISO\_08047, TISO\_11097 TISO\_16495, TISO\_27353, TISO\_37260, and TISO\_37631) were also found, containing the four main domains (up to 18 for KR in TISO\_08047). Except TISO\_37631, these sequences also have thioesterase, sulfotransferase, or peptide-synthesis-related domains, and thus they might be in charge of the synthesis of more complex lipids.

	-

**Figure 7.** Cluster of candidate proteins suspected involved in PKS PUFA synthesis pathway in *T. lutea*. The name of each protein is annotated with TISO\_ (for *Tisochrysis lutea*) and associated number. In the legend, the text written in bold italic correspond to domain names as shown in NCBI conserved domain database, followed by its suspected role.

#### **3. Discussion**

This study investigated long-chain PUFA synthesis pathways in the haptophyte *Tisochrysis lutea* using the incorporation of 13CO2. Addition of 13CO2 did not affect *T. lutea* physiology. Cell viability remained above 93% during the experiment, while cell complexity and chlorophyll content did not vary significantly according to sampling time. Cell size (as attested by FSC) increased slightly during the 24 h experiment. *T. lutea* produced FA to a level of 7% of POC; predominantly in the form of PL (66%).

Major FA of *T. lutea* were similar in proportions to those found in other prymnesiophycea (Haptophytes), i.e., 14:0, 16:0, 18:1n-9, 18:4n-3, and 22:6n-3 [41–45]. As reported before in Huang et al. (2019) [46], *T. lutea* had a low content of neutral lipids during exponential phase, and PUFA were mainly found in the polar fraction. *Tisochrysis lutea* accumulates neutral lipids mainly during stationary phase or under nutritive limitations [45].

The final level of atomic enrichment (AE) into the different FA witnessed active synthesis, as most fatty acids had a higher AE than that of POC (30% on average for the two balloons). 22:5n-6 was the most enriched long chain PUFA (LC-PUFA) in the PL fraction. 22:5n-6 and 18:2n-6 were the only 13C labelled n-6 fatty acids detectable by GC-c-IRMS. None of the known synthesis intermediates (18:3n-6, 20:3n-6, 20:4n-6, and 22:4n-6) between 18:2n-6 and 22:5n-6 [4] had measurable 13C-labelling and were below 1% in the FA profile during our experiment. It is then difficult to hypothesize the pathway used to create 22:5n-6 with this missing information. However, even though the different intermediates were undetectable, 18:2n-6 and 22:5n-6 atomic enrichments being very close cannot exclude them

to be related to each other. While studying the existence of an alternative Δ8 desaturase in Haptophyte, Qi et al. (2002) [8] noticed the absence of intermediates of the n-6 Δ8 desaturase pathway (20:2n-6, 20:3n-6 and 20:4n-6)) in *Isochrysis galbana*. It was attributed to relatively high active enzymes that could form the end-product 22:5n-6 with a rapid flow through these n-6 intermediates. Our results agree with this, as 13C enrichment of n-6 intermediates could not be detected by compound specific isotope analysis. To demonstrate the existence of these pathways, it would be interesting to combine functional analysis of desaturases by expression in yeast and GC-c-IRMS monitoring of the intermediates after 13C labelling of their precursors.

However, it is also possible that another pathway not involving "classical" n-6 FA intermediates exist in *T. lutea*. Previous studies showed the existence of PKS genes in various species of the prymnesiophytes including *Isochrysis galbana* [47], closely phylogenetically related to *Tisochrysis lutea*. We identified five candidates; proteins potentially involved in PKS synthesis pathway in *T. lutea*. Even if their function has not been verified, it is possible that at least one of the proteins presented in Figure 7 was responsible for the formation of n-6 PUFA in the haptophyte. Thus, our hypothesis is that an n-6 PKS pathway might also exist in *T. lutea* (Figure 8). Finally, PKS and "classical" n-6 routes might not be completely independent and could interact in the synthesis of 22:5n-6 in *T. lutea*.

**Figure 8.** Hypothesized pathways for 22:5n-6 synthesis in *T. lutea* in the PL. Numbers in the boxes correspond to final AE value. The triangles symbolize the desaturases (front-end in yellow and methyl-end in purple), the circles the enzymes involved PKS pathway (KR: 3-ketoacyl synthase, KS: 3-ketoacyl-ACP-reductase, DH: dehydrase, 2.2I: 2-trans, 2-cis isomerase, 2.3I: 2-trans, 2-cis isomerase, ER: enoyl reductase), and the squares the elongases.

Despite being one of the most abundant FA, 18:4n-3 showed a low 13C-enrichment (23%). The synthesis of 18:4n-3 from 18:3n-3 by Δ6 desaturase had already been described by *Isochrysis* sp. [48]. We assume that such activity also exists in *Tisochrysis*, phylogenetically close to *Isochrysis*. However, as 18:3n-3 co-elute with 18:1n-9, it was not possible to measure its AE and to assess whether this could be a limiting step in n-3 pathway (Figure 9). The 18:5n-3 had the lowest enrichment, and the ratio 18:5n-3/18:4n-3 was below the threshold value (R = 0.78), indicating a feasible transformation of 18:4n-3 into 18:5n-3. The existence of Δ3 desaturase that could support the production of 18:5n-3 (18:5Δ3,6,9,12,15) from 18:4n-3 (18:4Δ6,9,12,15) had been suggested by Joseph (1975) [49] to explain the presence of this unusual FA in dinophytes. A more recent study by Ahman et al. (2011) [23] showed in *Ostreococcus lucimarinus* that a Δ4 desaturase was surprisingly able to add a double bond in 18:4n-3 at the Δ3 position leading to the formation of 18:5n-3 when the gene was expressed in yeast cell and supplemented by 18:4n-3 as substrate. With our results and the discovery of Ahman et al. (2011) [23], we proposed that a Δ4 desaturase of *T. lutea* might be able to act as a Δ3 desaturase on 18:4n-3 to produce 18:5n-3 (Figure 9). Desaturation of 18:4n-3 into 18:5n-3 had been previously hypothesized by Kotajima et al. (2014) [50] in the prymnesiophyte *Emiliania huxleyi*.

**Figure 9.** Hypothesized pathways to produce 18:5n-3 in *T. lutea*. Numbers in boxes correspond to final mean AE value, and number in the yellow box the mean value of ratio of the two surrounding fatty acids. The triangles symbolize the desaturases (front-end in yellow and methyl-end in purple), the circles the enzymes involved PKS pathway (KR: 3-ketoacyl synthase, KS: 3-ketoacyl-ACP-reductase, DH: dehydrase, 2.2I: 2-trans, 2-cis isomerase, 2.3I: 2-trans, 2-cis isomerase, ER: enoyl reductase). The directions with dashed arrows cannot be proven with the enrichment dynamics.

The 18:5n-3 was also described as an intermediate of 22:6n-3 synthesis by PKS pathway [4]. However, its low enrichment, as compared to 22:6n-3, appeared not compatible with a hypothetical production through this pathway. Nevertheless, one may speculate that there are two separated PKS pathways, one for the 22:6n-3 and one for the 18:5n-3, as these two PUFA are localized in different cell compartments. The 18:5n-3 is generally associated with chloroplastic glycolipids, while the 22:6n-3 is predominant in the other cellular compartments [51–53].

Surprisingly, 20:5n-3 in PL was more enriched than 18:4n-3, its precursor in the n-3 pathway [4]. As AE of 20:5n-3 is higher than AE of 18:4n-3, it seems very unlikely that 20:5n-3 was produced via the pathway involving 18:4n-3 elongation and 20:4n-3 Δ5 desaturation. The existence of the alternative Δ8 desaturase pathway have been studied before in *Isochrysis galbana* and *Pavlova lutheri* [8,54,55]. However, as for the n-6 PUFA, intermediates (20:3n-3 and 20:4n-3) of the alternative Δ8 pathway were not detected by fatty acid analysis of *Isochrysis galbana* [8]. Similarly, in our study, intermediates (20:3n-3 and 20:4n-3) of this pathway to synthesize 20:5n-3 have not been found in sufficient amount to be measured by CSIA. As proposed by Qi et al. (2002) [8] for *Isochrysis galbana*, the synthesis of 20:5n-3 via 20:3n-3 and 20:4n-3 by *Tisochrysis lutea* might be very rapid, explaining why these two intermediates were only found in trace amounts (0.12% and 0.02% in PL and 0.05% and 0.33% in NL, respectively).

Due to their lower enrichments, 18:4n-3 and 18:5n-3 seemed unlikely involved in long chain PUFA synthesis such as 20:5n-3 and 22:6n-3. Based on the enrichment dynamics, elongation of 20:5n-3 into 22:5n-3 and further desaturation into 22:6n-3, respectively, by Δ5 elongase and Δ4 desaturase could be possible in *Tisochrysis lutea*. Ratio 22:5n-3/20:5n-3 in PL was within the threshold, indicating a simultaneous enrichment of both 20:5n-3 and 22:5n-3 in *T. lutea*. Such enzymes have been evidenced in haptophytes [54,56,57].

Considering the diversity of PKS gene in haptophytes [47], the possibility of production of 22:6n-3 directly by PKS PUFA synthesis pathway might be possible, as previously shown with thraustochytrids [10,58]. Synthesis of 22:6n-3 by PKS pathway might be at play in parallel with the n-3 pathway. Indeed, we identified a protein cluster gathering the four main domains potentially coding for the enzymes used in PKS PUFA synthesis pathways: ketoacyl reductase (KR), polyketide synthase (KS), dehydrase/dehydrogenase (DH), and enoyl reductase (ER). Protein clusters are groups of similar proteins that most likely shared the same or similar functions [59]. By considering this cluster (candidate proteins TISO\_14962, TISO\_14968, TISO\_14968, TISO\_14973, TISO\_14975, and TISO\_14977, Figure 8), it could be possible that these proteins act together and allow n-3 PUFA synthesis via PKS pathway. Interestingly, protein TISO\_14973, while possessing only two of the four domains of interest (KS and DH), presented a specific n-3 domain. This protein might act concomitantly with the other proteins of the same cluster and allow the access to the missing reductase activities (KR and ER). Finally, the ratio 22:5n-6/22:6n-3 was below the threshold value making possible the conversion of 22:5n-6 into 22:6n-3 if we assumed that ω3-desaturase might exist in haptophyte. Synthesis of 22:6n-3 by both n-3 and n-6 pathway might be feasible in *Tisochrysis lutea* (Figure 10). These different ways to produce 22:6n-3 might contribute to betaine lipids synthesis. Indeed, betaine lipids are generally highly unsaturated in C20 and C22 PUFA, especially in 22:6n-3 in haptophytes [60–63].

**Figure 10.** Hypothesized pathways to produce DHA in *T. lutea*. Numbers in boxes correspond to final mean AE value, and number in the yellow box is the mean value of ratio of the two surrounding fatty acids. The triangles symbolize the desaturases (front-end in yellow and methyl-end in purple), the circles the enzymes involved in PKS pathway (KR: 3-ketoacyl synthase, KS: 3-ketoacyl-ACP-reductase, DH: dehydrase, 2.2I/2.3I: 2-trans, 2-cis or 2-trans, 3-cis isomerases, ER: enoyl-reductase), and the squares the elongases.