Section 1 Pathophysiology

#### **Chapter 1**

### Role of Spiral Steroids in Pregnancy and Pre-Eclampsia

*Fred Chasalow*

#### **Abstract**

My laboratory discovered a new type of steroids. The structure of these steroids is unique in three ways: (i) they have 23, 24 or 25 carbon atoms – no other known vertebrate steroid has more than 21 carbon atoms; (ii) they are phosphodiesters – no other steroid phosphodiesters are known; and (iii) some of them have a spiral steroid at carbon 17 – no other endogenous spiral steroids are known. In total, our laboratory had elucidated the structure and path of biosynthesis for more than 20 related compounds. We have developed an LC–MS method and a MS–MS method to measure the compounds in small samples (< 1 ml). Synthetic compounds with similar spiral steroids (e.g., spironolactone) function as potassium sparing hormones but there were no known endogenous hormones with this function. We propose that the natural spiral steroids have that function. Endogenous compounds with these functions would have important roles in the physiology of pregnancy, pre-eclampsia, and eclampsia. This chapter will review the proposed physiology and pathology of the spiral steroids during pregnancy. There are many details to confirm but this is a useful paradigm.

**Keywords:** hypertension, proteinuria, hypokalemia, edema, spiral steroids, Ionotropin

#### **1. Introduction**

Here is a brief history of the milestones on the discovery path that led to the discovery of phosphodiester spiral steroids and the recognition of their function as potassium sparing hormones (KSH):


were DLM and all four were not detectable by 2 weeks of age. We proposed that SLO was an enzyme defect in a previously unknown pathway that produced a compound that was potassium sparing [4].


In summary, this chapter proposes a new paradigm to account for the symptoms of pre-eclampsia. The paradigm also accounts for the long-term increased risk of both cardiovascular disease and end-stage renal disease in affected women and their offspring [15, 16].

#### **2. Biochemistry of steroid phosphodiesters**

This section describes the biochemistry of steroid phosphodiesters. I have used Ionotropin as a key word in every paper about steroid phosphodiesters. I suggest other investigators do likewise.

Ionotropin was the name we assigned to the steroid phosphodiester that was present in human serum and not present in serum from infants with SLO syndrome. We now know that there are two compounds that fit the definition of Ionotropin – C339 and C341. C339 and C341 are both present in human blood and were not present in serum from an infant with SLO. This usage would be equivalent to using glucocorticoid as a hormone type name and cortisol and corticosterone as specific compounds.

#### **2.1 Symbol convention**

Based on the steroid fragment observed on mass spectroscopy, we assigned a symbol with four characters in the 'Zabc' format [11]. The Z identifies which phosphodiester is present in the molecule: (a) C = phosphocholine, (b) P = phosphoethanolamine and (c) X = unknown. The 'abc' identifies the mass of the steroid fragment observed in a positive ion mass spectrum. The method is not antibody dependent. Anyone with a mass spectrometer can identify the appropriate symbol for any steroid phosphodiester. Note that, potentially, there could be isomers that share the same mass ion and phosphodiester fragment.

C339, C341, E339, and E341 were all present in bovine adrenal extracts but neither E339 nor E341 were detectable in serum from any species that we tested. This observation points to adrenal cortex as the site of synthesis.

#### **2.2 Numbering convention**

When only phosphodiester steroids with 23 carbon atoms were known, it did not make much difference which carbon was designated # 22 or # 23. Both are part of the E-ring. However, when we recognized that the added carbon atoms were derived from acyl-CoEnzyme A, we have revised the numbering scheme to reflect their common origin (**Figure 1**).

#### **2.3 Mass spectrometric methods**

Two basic methods were used. The first method used LC–MS with Atmospheric Pressure Chemical Ionization (APCI) in the positive ion mode [9]. Voltages were

#### **Figure 1.**

*Structures of representative steroid phosphodiesters. Starting in the upper left and going counter-clockwise, the compounds are C313, C339, C351, and C381. Carbon 17 is the spiral steroid. Ring E is painted green and the extra side-chain carbons are painted yellow. C339 is shown with the original, classical numbering scheme [17]. The revised scheme is shown for both C353 and C381. The new scheme recognizes carbon 22 as the carboxyl carbon of the CoEnzyme A acyl group. This numbering scheme clarifies the proposed common origin of the extra carbon atoms.*

selected to minimize fragmentation. The second method used direct injection into a quadrupole ion trap mass spectrometer [12]. Spectra were collected both with and without an additional fragmentation voltage. MS–MS analysis was also used to confirm parent-fragment ion relationships.

Steroids have molecular masses in the range 270 to 400 Da. The smallest steroid fragment from a phosphodiester has a molecular mass of 297 Da and the largest fragment thus far identified has a mass of 413 Da.

#### **2.4 Trial and error (T&E) determination of chemical composition**

**Table 1** illustrates the use of the T&E method to propose a composition for C381. As shown on Line 7, only one composition of carbon, hydrogen, and oxygen atoms can form a molecule with a mass of 398 Da - C**25**O**4**H**34**. Readers are invited to test other molecular compositions to generate a molecule with a mass of 398 Da. Similar T&E tables have been used for each of the steroid fragments we identified. The observation that only one composition fits the mass may be a coincidence but it certainly was useful. Occam's razor suggests that the phosphodiesters are all related, as precursors and/or metabolites. If this is not true, then there must be other, as yet undetected, phosphodiester steroids.


*Line: Each line describes a trial of a possible composition.*

*Carbons: The number of carbon atoms in this specific trial.*

*Oxygen: The number of oxygen atoms in this specific trial.*

*C + O: The contribution of the carbon and oxygen atoms to the mass.*

*Hmax: Maximum number of hydrogen atoms – 2+ 2 for each carbon atom.*

*Hreq: Difference between m/z and "C+O".*

*m/z: mass of the steroid fragment plus 17 Da- the fragment has lost an OH.*

*Delta: the number of delta necessary to complete a molecule. Delta is ½ the difference between Hmax and Hreq. Delta is the number of rings and double bonds in the molecule. The basic steroid structure has four rings. Ring E contributes 3 delta – ring, alkene, and the carboxylic acid. Thus, delta must be 7 or larger.*

*Conclusion: Line 7 (in bold) shows the molecular composition is C25O4H<sup>34</sup> and delta must be 9.*

*Isomers for the proposed structure of C381 are not eliminated by the T&E analysis. The same analysis has been done for each steroid fragment.*

#### **Table 1.**

*Trial and error (T&E) analysis of composition of C381.*

#### **2.5 Spiral steroid biosynthesis**

All of the newly discovered compounds are either phosphocholine (PC) steroid diesters or phosphoethanolamine (PE) diesters. The presence of the choline phosphodiester was confirmed by **<sup>31</sup>**P-NMR (**Figure 2**) and by the presence of a characteristic fragment at m/z = 184 Da in mass spectra. In humans, both choline and ethanolamine may be essential nutriments. The phosphodiester could be added to a steroid by condensation with CDP-serine and subsequent decarboxylation (see **Figure 2**). Based on the phosphodiesters we have identified, the acceptor steroid seems to be 17α-hydroxy-pregna-5,7-dienolone. Shackleton has isolated this compound from patients with SLO [19] and Slominski has confirmed that enzymes exist to convert 7-dehydrocholesterol to the same precursor [20].

The working theory is that the extra carbons are added by condensation of C313 or E313 with an acyl CoA (**Figure 3**). The three most common CoA acyl groups are: (i) acetyl, (ii) propyl, and (iii) acetoacetyl. The three lead to steroids with 23, 24, and 25 carbon atoms, respectively (**Table 2**). The three carboxylic acid intermediates were identified by their mass spectra. We can identify compounds which have hydroxy groups by MS–MS fragmentation (by loss of 18 Da). However, it does not identify which specific carbon atom had been hydroxylated.

C341 is the major spiral lactone in adult serum with lessor quantities of C337 and C339. These compounds differ by stepwise reduction of the two alkenes in their common C313 precursor. For cholesterol biosynthesis, the Δ7–8 bond must be reduced first because cholesterol has a Δ5–6 alkene but not a Δ7–8 bond. The same enzyme could be responsible for the reduction of Δ7–8 alkene to reduce C337 to C339. A second reduction step is necessary to reduce the Δ5–6 bond. Although testosterone is reduced to form the 5α derivative, that enzyme substrate specificity requires a Δ4, 3-ketone. As the phosphodiester blocks the ketone at carbon-3, that enzyme could not reduce C339. There is a reductase that generates 5β-metabolites. It forms cholic acid for bile. Thus, an enzyme with this specificity would produce the 5β-C341 isomer. Note that digoxin is also a 5β steroid.

The takeaway lesson from **Figure 4** is that the 5β isomer would fit like a key into a specific binding site in which the 5α isomer would not fit. The stereo-specificity of C341 is probably significant because the major weak androgen in humans (but not in most other species) is DHEA-S, which is a 5α-steroid. If C341 were a 5α- steroid, then both DHEA and 5α-dihydrotestosterone could both interfere with its function by binding at the receptor for C341, whatever it might be. Recall that spironolactone also binds to both the androgen receptor and the KSH receptor. In fact, this cross-

#### **Figure 2.**

*Biosynthesis of steroid phosphodiesters. Left panel. <sup>31</sup>P–NMR of synthetic DHEA-phosphodiester [18] and of C341 obtained by isolation from bovine adrenal extracts [9]. The three peaks are caused by the three cations (H+, Na+, and K+). Right Panel. Condensation of serine-CDP with 17α-OH-pregna-5,7-dienolone [19, 20] to form E313. We do not know the order of the two reactions – Decarboxylation and esterification. Mass spectroscopy confirmed E313 was present in adrenal extracts [9].*

#### **Figure 3.**

*Synthesis of phosphodiester spiral steroid lactones. Starting with E313, this figure shows the formation of the spiral steroid lactones. There are several steps at the Blue arrow: N-methylation, Δ7–8 reduction, and condensation with acyl-coenzyme A. The lactone ring formation occurs at the Red arrow. The order of the steps has not been positively determined. The three boxes show how the three Acyl groups lead to the three different side chains at carbon 23. The conversion of PE compounds to PC compounds (N-methylation) may occur at any step.*

binding makes spironolactone a less desirable pharmaceutical. Chickens and turkeys do not use DHEA as a weak androgen. This may explain why their serum has C339, but not C341, as the major spiral lactone [21].

#### **2.6 Tissue specificity**

Question: why do we need all three classes of spiral lactones?

Answer: Tissue specificity. Pre-pubertal children only have 23-carbon lactones. Gonad extracts and serum from pregnant women have 24-carbon lactones. Milk and high K+ breast cysts have 25-carbon lactones. There are multiple forms of the NaK-ATPase. We need to isolate each of the forms and evaluate their binding constants to the different spiral steroids at the different forms of NaK-ATPase.

Question: why do need both PE and PC phosphodiesters?

Answer: Best answer at present is the PE compounds are for storage until needed. N-methylation is ACTH dependent. Thus, as part of the stress response the epinephrine increases glycolysis and the spiral lactone increases heart efficiency [10]. We suggest (without direct proof) that the same process occurs during childbirth.

#### **2.7 Summary of biochemistry**

The last discovery of a novel steroid was of aldosterone and that occurred in the 1950s. The general consensus has been that all of the steroids were already known. Hamlyn's claim to the discovery of endogenous ouabain has not been widely accepted [7]. They reported isolating 13 μg from 80 liters of plasma (o.2 ng/ml). Blaustein, one of his colleagues, has even published a paper asking, "Why is endogenous ouabain not more widely accepted?" [22]. Nicholls replied saying, "Ouabain, a circulating hormone secreted by the adrenals, is pivotal in cardiovascular disease,

#### *Role of Spiral Steroids in Pregnancy and Pre-Eclampsia DOI: http://dx.doi.org/10.5772/intechopen.100337*


*\*Compounds purified to near homogeneity.*

*@ Mass spectrum also identified phosphoethanolamine (Exxx).*

*! Spiral steroid lactone.*

*# Site of hydroxy unconfirmed. Likely possible sites are at carbons 11 & 16. Compounds with an extra hydroxy fragment by loss of water (18 Da). This eliminates hydroxy groups at the axial carbons 18, 19, and 21.*

*+Carboxyl compounds must be protonated in the positive ion spectrum.*

*¶ This fragment was detected in milk extracts from cows, sheep and goats. & This fragment was only detected in fetal calf serum extracts.*

#### **Table 2.**

*Steroid phosphodiesters identified by Mass Spectroscopy.*

#### **Figure 4.**

*3D images of C341, a spiral steroid. Color code: carbon – grey; oxygen - red; phosphorus – orange; nitrogen – blue. Hydrogen atoms are not shown in these images. From bottom to the top, the ring designations are A, B, C, D, & E. Panel A and B show two different views of the 5β stereoisomer of C341. Ring A and Ring E, the spiral ring, are both perpendicular to the plane generated by Rings B, C and D. Panel C shows the 5α stereoisomer. Note that in the 5α stereoisomer, Ring A, B, C, and D are co-planar and only the plane of Ring E is perpendicular to the plane of the four rings. In both stereoisomers, the PC fragment has free rotation around the steroid plane.*

fact or fantasy?" [23]. Nicholls described two criteria required for an endogenous hormone: [a] biosynthetic pathway and [b] a method of assay not dependent on antibody specificity. Endogenous ouabain satisfies neither criteria. In fact, Baecher developed an ultrasensitive LC–MS method to measure serum levels of "endogenous ouabain" down to less than 2 pg./ml and could find none [24]. This section describes both a biosynthetic path to the spiral steroids and methods to measure spiral lactones by mass spectroscopy. It is unclear what Hamlyn measured but it is time to consider the role of the spiral lactones as the real endogenous KSH.

#### **3. Physiology of spiral steroid phosphodiesters**

This section describes our knowledge of the function of spiral steroids. As spiral steroid phosphodiesters are also present in oysters, the function is not limited to mammals but is probably common throughout the animal kingdom [25]. Plants seem to use cardiotonic glycosides for the same function. Amphibians use marinobufagenin and related compounds as poisons to discourage predators [26]. We have not measured spiral steroids in amphibian serum to identify which spiral steroid is used in their internal physiology. Note that marinobufagenin can only be obtained from amphibian skin after extensive hydrolysis. As such, it would not be expected to be present in serum.

#### **3.1 Why do we need to regulate intracellular K+**

For creatures living in the sea, ocean electrolytes are 460 mM Na + and 10 mM K+. This ratio closely resembles the electrolyte ratio in plasma 145 mM Na + and 4 mM K+. In contrast, intracellular electrolytes are 10 mM Na + and 140 mM K+. Although we know about the role of mineralocorticoids to recover needed Na+, until 2016, there were no known mechanisms to maintain intracellular K+ levels or to recover K+ in the kidney.

Most plants and animals have high levels of both Na + and K+ in their tissues and/or fluids. Thus, there is little need for a concentration mechanism for life forms that have free access to environmental foodstuffs. However, *in utero*, fetuses only have access to maternal serum electrolytes via the placenta. The fetus must concentrate K+ about 20-fold and must maintain the intracellular levels, despite passive diffusion of K+ from a high K+ intracellular fluid to a low K+ extracellular fluid.

#### **3.2 Background to endogenous K+ sparing hormones or diuretics**

None known.

#### **3.3 Background to synthetic K+ sparing diuretics (KSD)**

There are two types of chemicals classified as KSD and they function by different mechanisms. The Steroid-type, represented by spironolactone, activates K+ transport by the NaK-ATPase. The AT type, represented by amiloride or triamterene, interfere with passage of Na + ions through the epithelial sodium channel (ENaC). This reduces the need to 'pump' Na + out of cells [27].

Steroid-type KSDs include: digoxin, ouabain, spironolactone, eplerenone, marinobufagenin. Common features include:

• E-ring lactone with 5, 6, or 7 atoms

*Role of Spiral Steroids in Pregnancy and Pre-Eclampsia DOI: http://dx.doi.org/10.5772/intechopen.100337*


Spiral steroid phosphodiesters have all four features.

AT Type compounds function by interfering with Na + passage through ENaC. This activity reduces the diffusion of Na + from high Na + extracellular fluids to low Na + intracellular fluids. This leads to lower intracellular osmotic pressure and 'spares' intracellular K+. The net affect is to generate a positive inotropic response [27].

#### **3.4 Potassium accumulation in human breast cyst fluids**

Earlier, because breast cysts were suspected of being precursors for breast cancer, the biochemistry of the cysts was investigated [5]. Based on electrolyte composition, there were two types. Type 1 had high K+ levels (60–100 mM) and Type 2 had potassium electrolyte levels resembling normal serum (5 mM). We investigated DLM levels in cyst fluid samples obtained in the normal course of patient care [6]. DLM was only present in the Type 1, high K+, samples and the levels were 10 times the levels detected in serum from normal women or men. We proposed that the basis for the high K+ levels was the presence of a K+ regulating hormone. Type 1 fluids were used to develop methods for extraction and chromatography. The new methods were different from that used to isolate 'ouabain' or 'digoxin' from plasma [7]. Doping experiments confirmed that the new method would not extract authentic cardiotonic glycosides. However, we could not collect sufficient Type 1 cyst fluid to purify the steroid phosphodiesters. In retrospect, it seems likely that C381 was the spiral steroid actually present in the Type 1 fluids.

#### **4. Biochemistry and physiology of spiral steroids during pregnancy**

Spiral steroids function as endogenous KSH and regulate both intracellular K+ levels and K+ recovery [27]. Regulation of K+ is particularly important during pregnancy because the fetus receives all of its nutrition via the placenta and does not have access to K+ rich foods (**Figure 5**).

#### **4.1 Fertilization**

After ova are fertilized, the cells divide and multiply. The growing cells need K+ for their intracellular fluids. We detected C369 and E369 in bovine ovarian extracts. C369 is a spiral steroid with 24 carbon atoms and a hydroxy group at an unidentified location (**Figure 6**). I propose that C369 is the spiral lactone that functions as KSH for fertilized ova. Both C329 and C353 are present in serum from pregnant women. C369 was present in serum from 10 out of 10 (5 males and 5 females) obligate heterozygotes for SLO [29]. At present, there is no explanation for the presence of C369 in serum from the heterozygotes but not in other men or women.

#### **4.2 Maternal spiral steroids during second trimester**

At 22–24 weeks of gestational age, there are five steroid phosphodiesters in maternal serum: C313, C329, C341, C353 and C381 (**Figure 7**). C313 and C329 have


#### **Figure 5.**

*Schematic regulation of potassium during pregnancy. The figure shows the proposed relationship between potassium and spiral steroids during pregnancy. Most of the processes are known, but the significance of the steroid phosphodiesters had not been recognized [28].*

#### **Figure 6.**

*Biosynthesis of C369. Red Arrows: condensation with Propyl-CoEnz A; Blue Arrows: Hydroxylation at unconfirmed carbon atom. C353 and C369 are both spiral steroids.*

*Role of Spiral Steroids in Pregnancy and Pre-Eclampsia DOI: http://dx.doi.org/10.5772/intechopen.100337*

#### **Figure 7.**

*Steroid phosphodiesters during the second trimester. Serum samples (22–24 weeks of gestational age, n = 20 normotensive women; n = 20 women with pre-eclampsia) were obtained from Global Alliance for the Prevention of Prematurity and Stillbirth (GAPPS). Left column: normotensive women; Right column: women with pre-eclampsia. Top: Z-scores based on intensity of miltefosine as internal control. Bottom: Representative mass spectra. Method of analysis: For each spectrum, the intensity of each ion was compared to the intensity of the ion generated by miltefosine (hexadecyl-phosphocholine). The mean and standard deviations of ions from the normotensive women were calculated and used to generate Z-scores for each of the 40 samples. The Z-scores for each sample were graphed as a cluster [12]. Ions are identified in Table 3.*

21 carbon atoms. The other three are spiral steroids with 23, 24, and 25 carbon atoms, respectively.

#### **4.3 Aldosterone signaling changes in the 3rd trimester**

During the third trimester, there is a so-called 'aldosterone-signaling defect.' In fact, there are actually two distinct aldosterone-signaling *changes* during pregnancy and these do not resolve until 2 weeks post-natal [30].

One change reduces the activity of the ENaC. This is equivalent to an AT type activity of KSD. This leads to Na + wasting in the fetal kidney and is a key, necessary step in producing the electrolytes for the amniotic fluid.

The second change is equivalent to the S Type of KSD, leading to increased activity of the NaK-ATPase pump. The increase leads to increased intracellular K+ in the fetal and maternal heart. The K-Ca- exchange mechanism increases calcium levels in the heart and results in a calcium-dependent, increased pressor response in both the fetal and maternal compartments.

The increased fetal pressor response is necessary because, as the fetus grows, the arterial resistance increases due to the increased length of the arterial bed. The increase in the maternal pressor response is needed because of the increased size of the vascular bed in the placenta. For both processes, the biology was known but the relationship to endogenous KSH was unknown because the existence of a KSH was unrecognized.

In summary, the aldosterone signaling changes are not a defect but are normal changes that are necessary during the second and third trimester.

#### **4.4 Preparation for milk production**

Milk is unique in that it is the only major extracellular fluid with K+ levels higher than Na + levels – 12-17 mM of K+ vs. 5–6 mM Na+ [31]. A KSH function should be necessary to concentrate K+ from plasma (4–6 mM) to the higher K+ levels in milk. In fact, milks from goats, cattle and sheep all had high levels of C381, suggesting that it is the KSH required to accumulate high levels of K+. This observation suggests that the NaK-ATPase isoform in breast tissue may be specific for C381, rather than for any other spiral lactone.

#### **4.5 Post-natal**

Post-natal, infants are fed milk, which is high in K+, and the need for a KSH ends but serum levels of spiral steroids remain detectable for about two weeks. Infants remain Na + wasting and usually lose about 10% of their birth weight. By two weeks of age, the need for KSH is over; the spiral steroids have been metabolized; aldosterone function is restored; Na + wasting ends; growth resumes [30]. Mother and infant "live happily ever after."

#### **4.6 Summary of the role of spiral steroids during pregnancy**

Spiral steroids, acting as KSHs, play a key role in K+ regulation during pregnancy. Ionotropin with 23 carbon atoms is the primary KSH for maternal function. The 24 carbon atom compounds, C353 and C369, function in the gonads and in the fetal-placental compartment. As the mother prepares for milk production, C381, the spiral steroid with 25 carbon atoms, directs the accumulation of K+. Ionotropin (C339 and/or C341) and C381 are DLM. C353 and C369 have the same spiral lactone epitope and are probably DLM, but we have not confirmed that suggestion by isolation and testing of extracts. All of these compounds are phosphocholine steroid diesters. The corresponding phosphoethanolamine steroid diesters are present in extracts from tissues that ordinarily synthesize steroid hormones but are only present in trace amounts (if at all) in serum [9].


*The identify of each steroid ion was confirmed by MS–MS analysis. All parent ions were Na + ions. C313 is the precursor for C341; C329 is the precursor for C369. One of the precursors was elevated in 11 of 19 samples from women with pre-eclampsia.*

*\* The ion detected is the steroid fragment after loss of the phosphocholine.*

*! The ion detected is derived from Na + ion after loss of trimethylamine (TMA).*

#### **Table 3.**

*Identification of phosphodiester steroids in serum (Figure 7).*

#### **5. Physiology of spiral steroids in pre-eclampsia**

Pre-eclampsia is a syndrome, not a disease [32]. As a syndrome, the diagnosis is made by hypertension and proteinuria. The symptoms can begin as early as 20 weeks of gestation [33–35]. For many patients with pre-eclampsia, there is little consequence during pregnancy. Monitoring and bed rest are often recommended. However, about 6–10% of affected women develop life-threatening hypertension and/or seizures. The only treatment is immediate C-section [15]. After C-section, the seizures and hypertension usually resolve.

In addition to the classical symptoms, during the third trimester, many affected patients also develop hypokalemia [36, 37]. In fact, there is a statistically significant (P < o.o5) inverse relationship between maternal serum K+ levels and maternal blood pressure [35]. Publications from 3rd world countries describe hypokalemia in patients with pre-eclampsia but publications from 1st world countries do not recognize hypokalemia as a symptom or risk factor.

There are several things to note in **Figure 7**.


We used three different statistical methods to evaluate the ion intensity of the C313, precursor of Ionotropin. First, we compared the mean and standard deviation of the of the samples from the normotensive women with the corresponding data from the women with pre-eclampsia. Second, we used Rank sum analysis. This method does not assume a normal distribution and is considered more robust than methods that imply a normal distribution. Third, we used the mean and standard deviation of the normotensive women to show Z-scores for all 40 samples. This set of data is presented in the clusters in **Figure 7**. There were 12 samples from the women with pre-eclampsia with Z-scores over 2 for either C313 or C329; there was only one sample from a normotensive woman with Z-score over 2. This distribution is statistically significant at the P < 0.01 level. With all three methods, the differences are statistically significant at the P < o.o5 level. Although there was an increase in the concentration of precursors, DLM was undetectable at this stage of gestation [39].

This data portends converting pre-eclampsia from a syndrome to at least two diseases. One disease, Type A, characterized by elevated levels of at least one of the spiral steroid precursors (either C313 or C329), a second disease, Type B, characterized by normal levels of the precursors. The takeaway lesson from this study is hypertension and proteinuria seem to be symptoms of more than one disease [40].

### **5.1 Proposed biopathology of pre-eclampsia**


The initial underlying biopathology seems to be inadequate placental implantation [41, 42]. Investigators have measured many, many hormones as possible risk factor or mediators, but none predict more than 35% of the patients who develop the symptoms, none predict hypokalemia, none predict risk of life-threatening hypertension [43–45], and none provide a biochemical basis for the increased lifelong risk of renal or cardiac disease.

#### **5.2 Significance of changes in aldosterone signaling**

If fetal K+ levels were inadequate, the placenta should synthesize spiral steroid precursors, C313 and C329 [12]. This leads to their increase in the maternal circulation. In turn, these compounds are converted to spiral steroids which function as KSHs. Elevated KSH has been documented in patients with pre-eclampsia as increased DLM levels. This seems to be a normal third trimester process occurring during the second trimester. The preeclampsia symptoms would be caused by interference in function of the ENaC in the kidney and by increased pressor activity in the heart due to the secondary increase in Ca++.

#### **Figure 8.**

*Changes in spiral steroids during pre-eclampsia. This figure integrates the regulatory process with the normal role of spiral steroids acting as KSH. The biology has been known. The underlying biochemistry was unknown prior to the discovery of the spiral steroids. The shuttling back and forth of steroid phosphodiesters between the mother and the fetal-placenta unit is similar to the synthesis and function of estriol.*

#### **5.3 Pilot study results**

C313, the precursor for C341, was detected in serum from pregnant women by the ion at m/z = 518 Da (generated by {a} 313 Da from the steroid fragment, {b} + 183 Da from the PC fragment, {c} + 23 Da from the Na+, and {d} -1 Da from the loss of the H+ = 518 Da.). 7 of 20 women diagnosed with preeclampsia had elevated levels (Z > 2) of C313 in serum collected at 22–24 weeks of gestation. Just like overdose of KSDs, elevated levels of C341 in maternal serum would be expected to lead to maternal hypertension, proteinuria and hypokalemia.

C329, the precursor for C369, was detected in serum from pregnant women by the ion at m/z = 475 Da. This ion is generated by loss of trimethyl amine (59 Da) from the Na + ion at m/z = 534 Da. 4 of 20 women diagnosed with preeclampsia had elevated levels (Z > 2) of C329. There was a statistically significant increase in concentration of C329 in the affected patients when compared to the normotensive control group. This would be expected to lead to increased levels of a KSD in the fetal circulation without corresponding increases in the maternal circulation. However, the incidence of samples with Z > 2 for C329 did not reach statistical significance; a larger sample size will be needed.

C329 is the precursor of C369. C369 was not detected in pre-pubertal children but was present in 10 of 10 obligate heterozygotes for SLO. There is no report of increased incidence of maternal hypertension or proteinuria in this group. Three of the 40 samples had high levels of C369, presumably associated with heterozygote carrier status for SLO.

A third group, 9 of 20 women with preeclampsia, had normal levels of both C313 and C329 at 22–24 weeks of gestation. There may be three different patterns: [a] high levels of C313 leading to maternal hypokalemia and life-threatening hypertension, [b] high levels of C329 leading to self-treatment of the fetal hypokalemia without generating maternal life-threatening hypertension, and [c] a 3rd group of patients with an unrelated origin of their symptoms. Overall, only about 5–10% of women with preeclampsia develop seizures and/or life-threatening hypertension later in pregnancy. The existence of 3 diseases sharing symptoms of proteinuria and hypertension might be the explanation for the lack of progress in developing therapy for these syndromes.

#### **5.4 Post-partum**

The green bars and peaks in **Figure 7** show the C381 levels in serum from pregnant women. At 22–24 weeks of gestation, only one of the 40 samples had elevated levels of C381, characterized by a score of Z > 2. There was no significant difference between the serum levels of C381 of normotensive pregnant women when compared to the serum levels of C381 from women with pre-eclampsia. C381 could stimulate milk production without affecting maternal heart or kidney function.

If during gestation, the mother had pre-eclampsia, long-term damage may have occurred due to persistent hyperspirolemia. Animal models treated with plant-derived cardiotonic steroids develop long-term heart and kidney consequences [46, 47].

#### **6. Therapy for pre-eclampsia**

#### **6.1 Failed therapies**

#### *6.1.1 Phosphodiesterase inhibitors*

One hypotheses is pre-eclampsia can be treated with phosphodiesterase inhibitors, including sildenafil citrate [48, 49]. However, Podymow writes, "As currently understood, the hypertension of preeclampsia is secondary to placental under perfusion, thus lowering systemic BP is not believed to reverse the primary pathogenic process." [50].

#### *6.1.2 Digibind*

Digibind is an FAB isolated from an antibody to digoxin and is used to treat patients with hypertension caused by digoxin toxicity [51]. As there are elevated levels of DLM in serum from women with pre-eclampsia, Digibind has been tested to determine if it would reduce hypertension in women with preeclampsia [51]. Infusion with Digibind does lead to a prompt decrease in blood pressure in affected women. However, the effect is short lived. Within 12 hours, blood pressure has returned to pre-therapeutic levels. The interpretation was that ane unknown agent was bound to the FAB and excreted. However, additional amounts were synthesized, leading to continued hypertension. The effort, if any, to confirm the identity of the unknown agent has not been published.

#### *6.1.3 Monoclonal antibodies to Marinobufagenin*

Marinobufagenin is a poison originally isolated from toad skin extracts [52]. Abi-Ghanem, with polyclonal antibodies, developed a chemifluorescent immunoassay [53]. Agunanne used the assay to confirm elevated marinbufagenin levels in women with preeclampsia [54]. Fedorova developed monoclonal antibodies and observed there was an unknown factor in serum of Dahl rats that was detected by their monoclonal antibody [55]. In the first publication, it was characterized as marinobufagenin-like, then as endogenous marinobufagenin [56]; most recently, just as marinobufagenin [52]. However, there are no publications describing characterization of marinobufagenin, or any plausible precursor or metabolite, from any mammalian source, other than by immunoassay.

Despite not knowing the true identity of the 'factor' detected by these antibodies, investigators have proposed a role for marinobufagenin in pre-eclampsia in women [57]. I do not doubt that there is at least one unknown substance that crossreacts with marinobufagenin-specific antibodies in serum from patients with preeclampsia. I doubt that it is marinobufagenin.

#### **6.2 Proposed therapy**

The Pilot Study showed increased levels of one of the spiral steroid precursors, C313 or C329, in the maternal circulation. The corresponding spiral steroids are C341 and C369. High levels of C369 were present in obligate heterozygotes with SLO but these women do not have pre-eclampsia [29]. Thus, the cause of hypertension and proteinuria would seem to be C341. This leads to two significant therapeutic suggestions: [1] monitor disease progression with C313 and [2] treat with C369 or its precursor, C329. The goal would be to stimulate KSH activity in the fetus without stimulating the function of a KSH in the maternal circulation.

#### **6.3 Proposed diagnostic method**

The pilot study was designed to maximize the chance of a clear positive response. In fact, statistically that was achieved. However, it is likely that the elevated level of C313 did not appear suddenly at 22 weeks of gestation. A large study is needed to determine when the elevated precursor levels begin and, later in gestation, which spiral steroids are elevated in the patients who develop eclampsia or HELPP syndrome [15].

#### **7. Conclusion**

One general theme in endocrinology is, "One disease to a customer." If all symptoms experienced by a patient are not explained by the proposed biochemistry, the patient has a syndrome, not a disease. This chapter title tells the story, "It's all about potassium." None of the reviews that I found recognize the significance of hypokalemia as part of the disease.

In detail, several facts stand out: [a] there is little evidence that pre-eclampsia is a single disease, [b] the common characterization of pre-eclampsia as a syndrome does not include hypokalemia, [c] without considering the role of spiral steroids, there is no recognized mechanism that shows how inadequate placental implantation leads to all of the classical symptoms of pre-eclampsia, hypokalemia or to the long-term increased risk of coronary or renal disease.

#### **Acknowledgements**

I specifically wish to recognize three very special colleagues: Dr. Ron Bochner, Dr. H. Leon Bradlow and Dr. Sandra Blethen (Chasalow).

Colleagues included, Dr. Kathryn King, LIJMC, Dr. Sharon Nachman, LIJMC, Dr. Gary Jarvis, VA Medical Center, San Francisco, CA and by Dr. Constance John, VA Medical Center, San Francisco, CA. Dr. John encouraged me and made laboratory space and equipment available. My two laboratory chiefs were Michael Davis and Lori Pierce-Cohen. Dr. Forbes Porter and Dr. Christopher Wassif of the NICHD provided serum samples from patients and obligate heterozygotes with Smith-Lemli-Opitz syndrome. Dr. Alisha Romano provided serum samples collected in the normal course of patient care.

Marvin Applets were used for drawing, displaying and characterizing chemical structures and reactions, Product Version 21.1 ChemAxon (https://www. Chemaxon.com).

#### **Funding statement**

This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. Dr. Ron Bochner personally funded the pilot study of women with pre-eclampsia. The Smith-Lemli-Opitz Foundation funded the investigation of the patients with SLO syndrome. AMUR Research Corp funded most of the original investigations. Kerix funded the purification of the compounds with 23 carbon atoms and the investigation of the chemical formulas. This work was partially supported by the Research Service of the United States Department of Veterans Affairs. Current support from IOMA LLC.

#### **Conflict of interest**

The authors declare no conflict of interest.

*Preeclampsia*

### **Author details**

Fred Chasalow

1 IOMA LLC, Belmont, CA, USA

2 VAMC, San Francisco, CA, USA

\*Address all correspondence to: fchasalow@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Role of Spiral Steroids in Pregnancy and Pre-Eclampsia DOI: http://dx.doi.org/10.5772/intechopen.100337*

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Section 2

## Factors Affecting Preeclampsia

#### **Chapter 2**

## Role of Vitamin D in Preeclampsia

*Simmi Kharb*

#### **Abstract**

Pathogenesis of preeclampsia involves immune dysfunction, placental implantation, abnormal angiogenesis, excessive inflammation, hypertension that may be affected by vitamin D. Human placenta expresses all the components for vitamin D signaling: Vitamin D receptor (VDR), retinoid X receptor (RXR), 1-alpha- hydroxylase (CYP27B1) and 24- hydroxylase (CYP24A1). Vitamin D binding protein plays a role in binding and transportation of 25 hydroxyvitamin D [25(OH)D] and 1,25(OH)2D3. Vitamin D is activated by 25-hydroxylase (CYP2R1) and 1-alpha -hydroxylase (CYP27B1) and is degraded by 24-hydroxylase (CYP24A1). Vitamin D supplementation is not recommended by WHO for pregnant women and allows recommended nutrient intake (RNI) of 200 IU (5 μg) per day. Further research requires serum 25(OH)D analysis and assessment of maternal and infant outcomes; pre-conceptional vitamin D status.

**Keywords:** Vitamin D, vitamin D receptor, cytochrome P450, pregnancy, preeclampsia, cord blood

#### **1. Introduction**

Pathogenesis of preeclampsia involves immune dysfunction, placental implantation, abnormal angiogenesis, excessive inflammation, hypertension that may be affected by vitamin D [1]. Preeclampsia complicates 2–8% of pregnancies globally and the incidence continues to increase worldwide. Preeclampsia (PE) is associated with significant maternal morbidity and mortality.

#### **2. Pathogenesis of preeclampsia**

Numerous pathophysiologic abnormalities have been suggested to explain the mechanisms of the origin of preeclampsia. Despite intensive research efforts, the etiology and pathogenesis of PE are not completely understood. The development of preeclampsia is influenced by genetic, immunologic, and environmental risk factors suggesting a multifactorial origin. Currently, there is no single reliable, cost-effective screening test for preeclampsia. A baseline laboratory evaluation is performed early in pregnancy in women who are at high risk for preeclampsia.

It is obvious that no single mechanism is responsible for this syndrome. The initiating abnormality is failed vascular remodeling of the vessels that supply the placental bed (stage 1). This was linked to the maternal syndrome of preeclampsia (stage 2). Two key features in the pathogenesis of preeclampsia are shallow endovascular cytotrophoblast invasion in the spiral arteries and endothelial cell dysfunction.

#### *Preeclampsia*

According to Barker's theory (also, called fetal programming or fetal origins of disease), origin of some adulthood chronic diseases such as cardiovascular diseases, hypertension and diabetes have their origin in intrauterine life. This hypothesis suggests that the intrauterine environment in which the fetus develops may be responsible for complications in adult life. Changes occurring in intrauterine environment and that somehow could disrupt normal development of the fetus can trigger metabolic changes, which may result in the development of long-term disorders. Preeclampsia has implications for future pregnancies and future cardiovascular risk.

#### **3. Vitamin D metabolism during pregnancy**

Since fetus completely relies on the maternal stores for its growth and development, vitamin D status during pregnancy has an important effect on this. During early pregnancy, 1,25(OH)2D increases and they continue to increase until delivery. This increase in 1,25(OH)2D is dependent on the available 25(OH)D levels and are independent of calcium metabolism (**Figure 1**).

The primary role of vitamin D in pregnancy is immunomodulatory in addition to its classical calcium regulatory function. According to Barker's hypothesis, the developmental origins of adult disease lie mainly in prenatal factors such as nutritional insults occurring during pregnancy and/or early infancy period [2].

Vitamin D metabolism during pregnancy and fetal development is different as compared with non-pregnant state, The conversion of vitamin D to 25(OH)D is unchanged during pregnancy. The conversion of 25(OH)D to 1,25(OH)2D during pregnancy is unique and unparalleled during life and at no other time during life 25(OH)D is so closely linked with 1,25(OH)2D production.

During pregnancy, the rise in 1,25(OH)2D in the mother and fetus is dependent on substrate availability i.e., 25(OH)D, and this is largely independent of calcium

**Figure 1.** *Overview of vitamin D metabolism, its role and mechanism of action.*

homeostasis. The 1,25(OH)2D serum concentrations double by 12th weeks of gestation and continue to rise two- to threefold from the non-pregnant baseline rising (to over 700pmol·L−1) attaining levels that would be toxic due to hypercalcemia to the non-pregnant individual, but which are essential during pregnancy. Neither in the mother nor in the fetus during the pregnant state, this conversion seems to be controlled by classic calcium homeostatic mechanisms. Calcium homeostasis, however, is not linked with this increase in 1,25(OH)2D, because there is no increase in calcium demand by either the mother or fetus at 12 weeks of gestation. In contrast, the increased 1,25(OH)2D levels remain sustained during pregnancy and during lactation these levels are not sustained when the maternal calcium demands are high.

The mechanism of uncoupling of calcium metabolism from 1,25(OH)2D generation during pregnancy and not lactation is not clear. It could be due to the fact that 1,25(OH)2D is an important immune modulator involved in maternal tolerance to the foreign fetus since pregnant women with preeclampsia have a clinical picture of inflammation and vasculitis, vitamin D deficiency has been implicated and vitamin D is a known modulator of inflammation [3].

Experimental animal studies have also strongly shown that vitamin D deficiency is a potential mechanism of placental dysfunction and respiratory maturation [4].

There is disruption of endothelial stability and an enhancement of "vascular leak" during preeclampsia and experimental animal models of preeclampsia have clearly demonstrated that endothelial instability leads to placental ischemia [5].

Vitamin D3,25(OH)D3 and 1,25(OH)2D3 stabilize endothelium and endothelium "leak" through non-genomic mechanisms and on equal molar basis, vitamin D3 has more potent action as compared to 25(OH)D3 or 1,25(OH)2D. Vitamin D3 is the most accessible form for cell membrane and it exists mainly bound to VDBP in circulation and only miniscule amount of vitamin D3 exist in the free form. Vitamin D3 has a longer half-life following its endogenous synthesis (in skin) as compared to the exogenous vitamin D taken orally and the half-life of 25(OH)D is weeks. Vitamin D3 when given at physiological doses of 4 000IU·d−1 or greater circulates in the "free" form at significant levels to be available to membrane insertion and subsequent endothelial stabilization that is likely to have profound effects on several disease processes. Recent studies have implicated maternal vitamin D deficiency as a risk factor for abnormal fetal growth patterns, adverse birth outcomes, increased risk of preterm birth. and reproductive failure [6, 7].

1,25-dihydroxyvitamin D [1,25(OH)2D] is primary bioactive form, and it does not readily cross the placenta, umbilical cord concentrations of its precursor, 25(OH) D, are similar to maternal concentrations. Placenta modulates circulating vitamin D metabolites in pregnant women and favors the uptake of DBP-bound 25(OH)D3 through a specific receptor system (LRP2-CUBN) and has CYP27B1 activity.

Both maternal decidua and fetal trophoblast have detectable CYP27B1 activity and they express VDR. Placental production of 1,25(OH)2D has been documented to be essential for immunosuppressive effects required for immune tolerance of implantation. Vitamin D may have a more extensive role in placental function, including trophoblastic differentiation and extravillous trophoblast invasion of the decidua and myometrium and a fundamental role in the process of conception, implantation and development of the placenta itself. However, the precise role of vitamin D in the process of implantation remains unclear.

Studies have shown that 1,25(OH)2D regulates homeobox gene HOXA10 expression in human endometrial stromal cells which is important for the development of endometrial development and implantation. Animal studies have shown that female rats on a vitamin D-deficient diet had overall reduction of fertility and failure of implantation and administration of 1,25(OH)2D corrected this.

#### *Preeclampsia*

In addition, vitamin D via its immunomodulatory actions may also influence implantation indirectly. Decidual synthesis of 1,25(OH)2D has the potential to influence uterine natural killer cells, dendritic cells, macrophages and T-cells throughout pregnancy, including inhibition of Th1 cytokines and promotion of Th2 cytokines, that have a significant role in the process of implantation [8].

Obesity is also a major contributing factor to vitamin D status in pregnant women that causes lowering of 25(OH)D levels in pregnant women with high body mass index (BMI).

#### **4. Vitamin D signaling in pregnancy**

Vitamin D signaling is important for normal placental function and fetal growth. Vitamin D maintains healthy cellular functions and redox and Ca2+ signaling systems and increases expression of both Nrf2 and the anti-aging protein Klotho, a major regulator of Ca2+ and redox signaling. Declining vitamin D levels reduces the stability of this regulatory signaling network and may cause many of the major diseases linked to vitamin D deficiency which are associated with a dysregulation in both ROS and Ca2+ signaling [9].

Also, vitamin D signaling depends on availability and turnover of active vitamin D receptor (VDR) ligand 1,25-dihydroxycholecalciferol and efficiency of VDR transactivation. Net availability of active hormone depends on the delivery of substrate and the balance of activating and inactivating enzymes, mainly secosteroid metabolizing p450 enzymes (e.g., various hydroxylase enzymes: 25 hydroxylase, 24 hydroxylase and 1- alpha hydroxylase). Out of these hydroxylases, 1- alpha hydroxylase is expressed in kidney and released in systemic circulation to serve as a critical activating enzyme in circulation. It is also synthesized in target tissues and activates local secosteroid. 1- alpha hydroxylase in kidney is upregulated by low calcium intake and parathyroid hormone inactivates both phosphatonins [10] as well as proinflammatory signal transduction downregulates its expression.

Transactivation of VDR depends on exact molecular structure, nuclear translocation, and presence of heterodimer retinoid X-receptor (RXR) and other nuclear cofactors to regulate gene expression, however, membrane receptor for these effects is not yet identified.

Rickets is a syndrome of impaired vitamin D signaling due to vitamin D3 deficiency and can be caused by inherited defects of the cascade, nutritional deficits, lack of sunlight exposure, malabsorption, and underlying diseases like chronic inflammation. Vitamin D signaling is complex and modulated at multiple levels.

1,25 (OH)2 D can diffuse freely across the plasma membrane and binds its highaffinity nuclear receptor (VDR, vitamin D receptor) to mediate its effects transcriptionally and post transcriptionally. In the transcriptional pathway, 1,25(OH)2D bind to VDR and forms a heterodimer complex (VDR- RXR complex) with retinoid X receptor (RXR). The VDR-RXR complex binds to vitamin D response element (VDRE) in the promoter region to regulate the target expression of vitamin D. Also, there is non-transcriptional pathway of vitamin D signaling having modulatory effects via binding of calcitriol- VDR complex with caveolae to stimulate signaling cascades namely, protein kinase C and mitogen-activated protein kinase. These signaling cascades regulate various cellular functions such as proliferation, differentiation, invasion, and apoptosis. Altered VDR expressions have been associated with various cancers, however, role of VDR and vitamin D signaling in pregnancy is poorly understood.

#### **5. Vitamin D- induced genomic alterations during pregnancy**

Vitamin D supplementation during pregnancy appears to affect genetic information of several highly functional modules related to systemic inflammation and immune responses and implicates the emergence of a distinctive immune response in women destined to develop preeclampsia [11].

Both non-genomic and genomic actions of vitamin D can affect epigenetic regulation of fetal development, and dynamic changes occur in epigenetic markers namely, methylation, hydroxylation, post translational modifications (covalent modifications) various short and long RNAs that regulate the transcriptional gene activity during the acquirement of specific cellular functions. A subset of epigenetics are programmed during early pregnancy that are stably maintained into adulthood [12].

#### **6. Role of vitamin D binding protein (VDBP) in PE**

VDBP is plasma carrier protein that binds metabolites of vitamin D to be transported in the body. Vitamin D-binding protein [VDBP, group-specific component (GC) of serum (GC-globulin)] is encoded by the GC gene. VDBP is synthesized mainly in liver and synthesized in adipose tissue, kidneys, and gonads. VDBP is 58 kDa glycosylated alpha-globulin composed of 458 amino acid residues in length and it folds into a triple-domain structure bound by disulphide bonds.

VDBP has immunomodulatory properties and is involved in chemotaxis of fatty acids and endotoxins. Immunological role for VDBP in pre-eclampsia in VDBP of placental origin has been documented as autoimmune target of autoantibodies in the sera of pre-eclamptic women compared with the sera of healthy non-pregnant women. Maternal obesity is associated with adverse health effects for both mother and newborn along with increased inflammation seems to be an important pathological mechanism for detrimental effects of obesity during pregnancy. However, role of vitamin D in the process is still remains to be clarified.

VDBP-macrophage activating factor (DBP-MAF) is involved in bone metabolism. VDBP has been shown to increase drastically during pregnancy as compared to non-pregnant women, reaching their peak in early third trimester and with the lowest level at approximately 36 weeks gestation. This increase is associated with increased total 25(OH)D and decreased free and bioavailable 25(OH)D to increase the capacity to store and metabolize more vitamin D to maintain sufficient concentration of vitamin D throughout pregnancy and lactation to support their increased requirements.

The increase in VDBP during pregnancy could also occur in response to rising estrogen. VDBP has been reported to increase when oestrogens levels are increased in conditions such as high stress states, some ovarian tumors and hormone replacement therapies.

Fetus obtains its supply of vitamin D via placenta which has also been shown to express VDBP. Placental cells express the components of vitamin D signaling including VDR and VDBP and can synthesize and respond to 1,25(OH)2D3 and 24,25(OH)2D. The maternal vitamin D compounds may enter the placental cells by endocytosis of 25(OH)D-VDBP and/or by diffusion of the free hormone to be transformed into 1,25(OH)2D3 or 24,25(OH)2D, however, the exact mechanism is not known. Without VDBP maternally derived 25(OH)D may not enter placental cells and its transformation into the active form of vitamin D and its transport to the fetus for utilization would not be possible.

#### *Preeclampsia*

SNPs of three genes involved in vitamin D metabolism including GC have been implicated in pre-eclampsia risk. GC-1 phenotype has been identified as a genetic marker for early detection for women at risk of pre-eclampsia. This has been shown that in South African (HIV endemic region) pregnant women complicated by pre-eclampsia that two SNPs of GC gene (rs4588 and rs7041) are more frequently present.

Status of VDBP and total 25(OH)D in preeclampsia is still not clear. Few studies have reported that different VDBP plasma concentrations in women who developed pre-eclampsia as compared to pregnant normotensive controls and no correlations have been noted between VDBP and total 25(OH)D. The increased oxidative stress in pregnancy may be responsible for the altered concentration of VDBP and vitamin D metabolism in placentae in preeclampsia. Moreover, proteinuria in preeclampsia have been shown to cause urinary loss of VDBP as compared to normotensive pregnancies possibly due to disruption of vitamin D metabolism and function through reduced VDBP.

Current evidence suggests that VDBP has been implicated in pregnancy, but its exact role is not yet fully understood. More focused studies are needed to address these limitations to disentangle the functions of VDBP and to clarify its role as a measure of vitamin D status and an important novel biomarker of pregnancy and reproductive outcomes.

#### **7. Role of cytochrome P450 in PE**

Two hepatic P450 enzymes catalyzing 25-hydroxylation of vitamin D3 (VD3) exist in mammalian liver namely, mitochondrial, and microsomal enzymes. Mitochondrial vitamin D3 25-hydroxylase is apparently identical with CYP27A.

VD3 is activated to 1α,25-dihydroxyvitamin D3 (1,25-D3) by cytochrome P450 2R1 (CYP2R1)/CYP27A1 and CYP27B1 (1-alpha-hydroxylase) sequentially and deactivated by multiple enzymes including CYP3A4. 1,25-D3 can activate the transcription of CYP3A genes. Activated vitamin D receptor (VDR) forms a heterodimer with retinoid X receptor α (RXRα) to recruit co-activators and translocate this to the nucleus for its binding to specific vitamin D responsive elements (VDRE), and thus activates the gene transcription. This transactivation effect modulates the nutrient bioavailability and drug metabolism. Also, extrarenal expression of CYP27B1 (1-alpha-hydroxylase) generates 1,25(OH)2D in numerous target tissues including the placenta and brain. Vitamin D receptor (VDR) regulates cytochrome P450 3A (CYP3A) expression in human and VDR-response elements are found in the promoter region of CYP3A genes [13].

#### **8. Vitamin D supplementation in pregnancy**

Vitamin D dysregulation during pregnancy has been linked to adverse effects on placental function and pregnancy and there is requirement for adequate vitamin D status across gestation. Pregnant women are at high risk of vitamin D deficiency (VDD) and VDD during pregnancy is associated with increased risk of gestational diabetes and preeclampsia. Since preeclampsia can affect offspring health resulting in low birth weight, poor skeletal health, impaired brain development, autoimmune disease, obesity, and insulin resistance.

Randomized controlled trials investigating vitamin D supplementation during pregnancy have revealed that increased vitamin D supplementation decreased complications of pregnancy and C-section births and improve birth outcome data.

#### *Role of Vitamin D in Preeclampsia DOI: http://dx.doi.org/10.5772/intechopen.100139*

Recent randomized controlled trials involving vitamin D supplementation in high-risk pregnancies have demonstrated decreased cesarean section rate and maternal hospitalization, decreased macrosomia and hospitalization in newborns of women with gestational diabetes. Favorable effects on insulin metabolism parameters, serum HDL cholesterol and total cholesterol concentrations in women with pre-eclampsia risk factors were also reported [14].

#### **9. WHO recommendations**

Vitamin D supplementation is not recommended for pregnant women to improve maternal and perinatal outcomes [15].

*Remarks:*


\*This is an extract from the relevant guideline (https://www.who.int/ publications-detail-redirect/9789240008120).

#### **10. Future research**

The complexity of vitamin D metabolism and functions involved in placental development are still to be fully elucidated and they are likely to be a key component of future studies of vitamin D in pregnancy. Further studies of vitamin D and adverse events in early pregnancy are required.

This needs to be clarified in future studies that how variations in vitamin D system in placenta and fetal trophoblast cells can affect implantation and regulate maintenance of a successful healthy pregnancy.

Role of vitamin D in maternal obesity is still not clear. Only a limited number of reports of vitamin D deficiency and miscarriage are available, and such studies need to be expanded by including more rigorous supplementation trials.

The mechanism of alteration of offspring epigenetic status by maternal VDD and the physiological impact of these epigenetic modifications remains uncertain. Future studies are needed to elucidate the mechanism and searching the windows for effective timely intervention via supplementation. Since VDD critically affects developmental programming of short- and long-term offspring metabolic and

neurobehavioral health, potentially via epigenetic mechanisms, exploration of mechanisms of non-genomic or genomic effects of vitamin D is required.

### **11. Conclusions**

A proper understanding of causal mechanisms that lead to adverse health in offspring born to VDD mothers is required for early diagnoses and improving treatment during pregnancy so as to prevent later adverse DOHaD (developmental origins of adult disease) effects in at-risk offspring and mothers in future. Some genetic variants of VDBP have also been reported to be associated with these adverse outcomes. Further studies are required to explore more accurate VDBP assays and exploring ethnic variation and potential confounders are needed to clarify whether VDBP is associated with reproductive health and pregnancy outcomes, and the mechanisms underlying these relationships and possible role of vitamin D during pregnancy to prevent adverse fetal and maternal outcome.

### **Acknowledgements**

Special thanks to my teachers, students and patients for inspiring me.

### **Conflict of interest**

None. There is no conflict of interest.

### **Notes/thanks/other declarations**

None.

#### **Author details**

Simmi Kharb

Department of Biochemistry, MRU, Pt. B.D. Sharma PGIMS, Pt. B.D. Sharma UHS, Rohtak, Haryana, India

\*Address all correspondence to: simmikh@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### *Preeclampsia*

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