OPEN ACCESS Research Article Spiral Phosphocholine ...(Gallus gallus domesticus) Citation: Fred...

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Research Article

Spiral Phosphocholine Steroids and DLM in Chicken Eggs (Gallus gallus domesticus)

Citation: Fred Chasalow. “Spiral Phosphocholine Steroids and DLM in Chicken Eggs (Gallus gallus domesticus)”. EC Paediatrics 8.12 (2019): 01-12.

*Corresponding Author: Fred Chasalow, Managing Partner IOMA LLC, Belmont, California, USA.Received: October 18, 2019; Published: November 11, 2019

AbstractChicken eggs can be separated into two separate fluids, commonly described as egg whites and egg yolks. There is a characteristic

difference in electrolytes between the two. Egg yolks have about 19 mg of potassium and 8 mg of sodium ions while the whites have 70 mg of potassium and 55 mg of sodium. Our theory is that the accumulation of the potassium ions is generated by a phosphocholine spiral steroid that functions in a manner similar to that of synthetic potassium sparing diuretics.

After separating whites from yolks, extracts were prepared with acetonitrile, filtered and analyzed by electrospray Tandem mass spectrometry. Each parent compound was detected as both Na+ and K+ cations, the proportion depending on the local electrolyte levels. The corresponding H+ cation had very low ion intensity. The egg white extracts contained three major phosphocholine esters: designated C361 > C339 > C337. These three compounds are steroids with 23 carbon atoms. C337 and C339 are spiral steroids and, earlier, had been isolated from both bovine and human serum. C361 was not detected in bovine or human serum but was isolated from bovine adrenals. The egg yolk extract had only one major phosphoester, C313.

We propose that the association of C337 and C339 with potassium accumulation in the egg whites is the result of their proposed function as potassium sparing hormones, perhaps by direct interaction with NaK-ATPase.

Previously, C337 and C339 were both identified as possible digoxin-like materials (DLM). To qualify as an endogenous DLM, an endogenous biosynthetic path is needed. Based on the compounds we found in the eggs, we propose a biosynthetic path that ac-counts for all of the phosphocholine compounds we found in both egg whites and yolks. These observations support our hypothesis that the steroid spiral lactones are the long sought DLM(s). Finally, egg white and yolks offer a good source for these compounds for investigation of function.

Keywords: Ionotropin; Phosphocholine steroid esters; DLM; Spiral Steroids; Potassium Sparing Hormone; Orphan Ligands

Fred Chasalow1,2*1Managing Partner IOMA LLC, Belmont, California, USA2Visiting Professor Dept. of Laboratory Medicine, VAMC, San Francisco, California, USA

AbbreviationsNa+: Sodium Cation; K+: Potassium Cation; RI: Relative Intensity; DLM: Digoxin-Like Materials; DHEA-S: Dehydroepiandrosterone-sulfate; MS: Mass Spectrometer; MSN: Multiple Staged MS Analysis; C313: Phosphocholine Ester of 3β, 17α-dihydroxy-pregna-5,7-dien-20-one

Highlights• C313 is the major PC-steroid in egg yolks.

• Egg whites have C337 and C339, which are spiral steroid lactones.

DOI: 10.31080/ecpe.2019.08.00593

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• C337 and C339 can’t act via nuclear receptors because there is no DNA in egg whites.

• Egg whites also have large amounts of C361, a metabolite of C313.

• Description of the biosynthetic pathway to the spiral steroid lactones.

Phosphocholine steroid conjugates are designated as Cxyz where C identifies the compound as a phosphocholine ester and xyz indi-cates the mass ion (M-17 Da, resulting from loss of 3β-hydroxy group) derived from the steroid fragment.

IntroductionThis is the 6th paper in the series describing the discovery of a new class of steroids and exploring their function. The new class has two

characteristics: (1) they are phosphocholine esters and (2) they are spiral steroids or possible precursors. A spiral compound is defined as a compound in which one carbon atom is part of two rings. Throughout this paper we use the naming convention we used for investiga-tion of phosphocholine esters in pre-eclampsia {Cabc}. The letter {C} indicates a compound is a phosphocholine ester and the three digits {abc} indicate the observed m/z value of the steroid fragment [2].

We first detected phosphocholine steroids as digoxin-like materials (DLM) [1]. In brief, we isolated three phosphocholine steroid conjugates with 23 carbon atoms that were also quantitated as DLM. The three compounds, C337, C339 and C341, are phosphocholine esters of spiral steroids with a γ-lactone ring. The compound with the most similarity to both spironolactone and digoxin, C341, we named Ionotropin. Based on the structural similarity, we propose that Ionotropin functions as a potassium sparing hormone [3]. It is our theory that the spiral steroid phosphoesters are the endogenous DLM. In addition to the three compounds with 23 carbon atoms, there was also a phosphocholine ester steroid with 21 carbon atoms, C313, 3β, 17α-dihydroxy-pregna-5,7-dien-20-one. Shackleton identified this ste-roid in patients with Smith-Lemli-Opitz syndrome, 7-dehydrosterol reductase deficiency [4]. The phosphocholine ester of this compound seems to be the precursor for the spiral steroids.

The first sources for the phosphocholine steroids were infant serum from patients with Smith-Lemli-Opitz syndrome [5] and from hu-man breast cyst fluids [6]. Both of these fluids were rich in DLM compounds, but it was difficult to get enough of either fluid to isolate the compounds. We screened many tissues for DLM. One of the fluids tested was chicken eggs. Egg whites, but not yolks, had DLM(s). With new methods for extraction and MS-MS analysis [2], this study sought (a) to confirm that spiral lactone phosphocholine esters were pres-ent in egg whites and (b) to confirm their absence in egg yolks.

This paper presents several significant findings:

• The major phosphocholine ester in egg yolks is C313, which is neither a DLM nor a spiral steroid.

• In contrast, egg whites had large amounts of both C337 and C339. These compounds are spiral steroids and are DLM. They differ by two Da, presumably the reduction of the Δ7-8 double bond.

• The concentration of C337 and C339 (~5 µM) in egg whites, gives an indication of its affinity to a binding protein. Although we don’t know the identity of the binding site, general biochemistry principles suggests that, if the affinity and serum levels were not similar, then regulation could not be achieved by variation in levels because the binding sites would either be mostly occupied or unoccupied.

• There is also a novel compound (C361) in egg whites that is not present in mammalian serum. Its structure suggests it is neither a precursor nor a metabolite of the spiral steroids and its function is obscure.

• Finally, we propose a role for a coenzyme A condensation in the biosynthetic pathway. The recognition of the pathway supports our theory that the spiral steroids are the long-sought endogenous DLM.

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MethodsWe purchased eggs (3 white eggs and 3 brown eggs) at a local farmer’s market. Store-bought ‘fresh’ eggs may have been laid more than

30 days prior to purchase. The eggs used in this study were purchased on a Saturday morning. The farmer reported the eggs were col-lected the previous Thursday. Extracts were made on Monday morning. Freshness of each egg was confirmed by “floating.” The whites and yolks were separated by typical cooking methods. Each fluid was extracted with 4 volumes (v/v) of acetonitrile. Brown egg extracts were kept separate from white egg extracts. Each extract was homogenized, centrifuged, and denatured proteins were removed. The extracts were stored at -80 C until analyzed. For analysis, 10 µl of a 0.2 mg/ml of miltefosine (Cayman Chemical, Ann Arbor, MI) prepared in 1:4 water/acetonitrile was added to 1 ml of extract. This process generated an internal standard (5 µM) to be used to evaluate the endogenous phosphocholine esters. For analysis, the extracts were filtered with Whatman Syringe filters with 0.2 µm pore size and analyzed by direct injection into the electrospray source of an LTQ-XL quadrupole ion trap mass spectrometer (MS) (Thermo Scientific, San Jose, CA). Flow rate was 10 µl/min. The capillary temperature was 275°C. Spray voltage was 4.4 volts. Spectral data was collected and averaged for 1 minute. Each sample was also fragmented with a 60 volt field. Finally, MSN analysis permitted fragmentation and analysis of specific ions from the ion trap. All spectra were generated in the positive ion mode

Quantitation

On the assumption that, on a molar basis, both miltefosine (hexadecyl-phosphocholine) and the steroid phosphocholine esters gener-ate similar ion intensities, the estimated concentration was based on the RI compared to miltefosine standard - 5 µM. Thus, if the RI of a particular phosphocholine ester ion was equal to the Intensity of miltefosine, then the concentration would be estimated as equal to 5 µM. As extracts had been prepared by mixing with 4 volumes of acetonitrile, if the RI was 20% then the estimated original concentration would be 5 µM. As the observed RI was about 20% of intensity of the miltefosine internal standard, the concentration in the egg white extract was 3 - 5 µM. For comparison, in young adults, the normal serum level of DHEA-S is 10 - 20 µM and for 17α-hydroxy-progesterone is 3 - 5 nM. When we have pure material, we may be able to verify the estimated concentration.

Results

Egg yolks: Figure 1 shows the mass spectra generated by egg yolk extracts: Panel A (Left)- brown eggs and Panel B (Right) white eggs (See table 1). Both white egg yolk and brown egg yolk extracts showed large intensities of both the K+ and Na+ cations of C313 but there were slightly higher levels of the K+ cation at m/z=534 Da. At much lower intensities, the same pattern was also observed for the spiral steroids, C339 and C341. The K+ ions of the spiral steroids C339 and C341 were both more intense than the Na+ ions. This confirmed that, in egg yolks, the K+ ion was present in higher concentrations then the Na+ ion. However, the relative amounts of the two spiral steroids changed. The white egg yolks had more C339 than C341. The brown egg yolks had more C341 than C339. This difference applied to both the K+ ions and the Na+ ions. The enzyme catalyzing the conversion of C339 to C341 is 5-dehydrosterol reductase. This enzyme also catalyzes the formation of cholic acid. Note that cholic acid and digoxin are both 5β sterols. Not much is known about the substrate specificity or regulation of this enzyme. It should be noted that 5α steroids are synthesized by reduction of Δ4-3 ketosteroids, such as testosterone [7].

Egg whites: Figure 2 shows the mass spectra generated from egg white extracts: Panel A (Left)- brown eggs and Panel B (Right) white eggs (Summarized in table 2).

Each spectrum shows six major peaks (the Big Six). The Big Six are actually three pairs. The elements of each pair are separated by 16 Da. The difference in mass between Na+ and K+ is 16 Da. Thus, in each pair, the one with the lower mass is the Na+ ion and the one with the higher mass is the K+ ion. The M+H ion is also present, but it is a minor peak. For the white eggs, all three pairs have about equal intensity, indicating about equal concentration of Na+ and K+. In contrast, for the brown eggs, in the three pairs, the Na+ ions have about twice the

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Structure of spiral steroids and precursors: (A) C313; (B) C337; (C) C339; (D) C341; (E) C361; (F) C361a; (G) C363.Notes: The MS was operated in positive ion mode. The hydroxy at carbon 3 is part of the phosphocholine ester. C313 is not a DLM. C337, C339 and C341 are spiral steroids and are DLM. The spiral structure is centered at carbon 17. The steroid fragments from C361, C361a and C363 could only be detected if they were protonated. The two added protons and the open E-ring account for the 20 Da difference between C341 (Panel D) and C361 (Panel E) or C361a (Panel F). Panel G shows the proposed structure of C363. Note that the difference between C341 and C363 is 22 Da. As there is only one reduceable bond in either C361 or C361a, the C20-C23 alkene must be reduced in C363, as shown in Panel G.

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Proposed steps in the biosynthesis of spiral steroids.

To allow for the extra carbons of C369 and C385, we have chosen to designate the added carbons starting with the carbon attached to CoEnzyme A as carbon 22 and proceeding to higher numbers from that starting point. Panel H Complex: R1 is Coenzyme A. R=H for steroids with 23 carbons. The consation product is shown in Panel J C375.Panel J: Structure of proposed CoEnzyme A intermediate complex. C375 is a branch point. Dehydration of the 20-hydroxy group leads to formation of C357 as shown in Panel L. if the C20-C23 alkene is reduced, as shown in Panel K, the spiral lactone cannot be formed because the side chain is not planar. The reduction is similar to the process that leads to long extension in fatty acid biosynthesis. Panel K: Reduction of the Δ7-8 alkene by 7-hydrosterol reductase leads to C361 (Panel E). A second reduction by 5-dehydrosterol reductase leads to C363 (Panel G). Panel L: The side chain of C357 is planar. This brings the two components of the lactone in close juxtaposition and permits formation of the spiral lactone C337 (Panel B).Panel M: C381 was identified in serum from pregnant women with pre-eclampsia. The numbering scheme is shown [2].Panel S: Spironolactone. Compare the structure of the two rings centered on carbon 17 to the similar structure in Panels B, C, D and M. Both rings are almost planar but the planes are perpendicular.

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Figure 1A: Left Panel: MS Spectrum from brown egg yolks. Figure 1B: Right Panel: MS spectrum from white egg yolks.

m/z Cation M+1 # C atoms Steroid Frag Symbol Composition518 Da Na+ 496 Da 21 313 Da C313 C21H3003

534 Da K+ 496 Da 21 313 Da C313 C21H3003

560 Da K+ 522 Da 23 339 Da C339 C23H3203

562 Da K+ 524 Da 23 341 Da C341 C23H3403

Table 1: Phosphocholine steroid esters in egg yolk extracts. The Na+ ions associated with C339 (544 Da) and C341 (546 Da) were both present but were not specifically marked on the figure.

Peak # m/z Assignment Steroid Formula Panel Proposed Structure1 542 Na+ ion of C337 354 Da C23H3003 B Δ5-Δ7 spiral steroid2 544 Na+ ion of C339 356 Da C23H3203 C Δ5- spiral steroid

3 558 K+ ion of C337 354 Da C23H3003 B Δ5-Δ7 spiral steroid4 560 K+ ion of C339 356 Da C23H3203 C Δ5 - spiral steroid5 566 Na+ ion of C361 378 Da C23H3804 E E-ring not closed6 582 K+ ion of C361 378 Da C23H3804 E E-ring not closed

Table 2: The masses and assignments of the 6 major ions in egg white extracts (ordered from left to right) as shown in figure 2. Note: All three of these compounds have 23 carbon atoms in the steroid.

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Figure 2A: Left Panel MS Spectrum. Extract from egg whites from brown eggs.

Figure 2B: Right panel MS Spectrum. Extract from egg whites from white eggs.

intensity of the corresponding K+ ion, indicating a much lower concentration of the potassium ions. Thus, there is a difference in electro-lytes between brown eggs and white eggs.

The egg whites from the white eggs have almost double the intensity of the C339 ions when compared to the intensity of the C337. The egg whites from the brown eggs have about equal intensity of C339 when compared to the intensity of C337. Putting these two observa-tions together, whites from white eggs, when compared to whites from brown eggs, the ratios of the intensity of [a] C339 to C337 ions and [b] K+ to Na+ ions are higher in white eggs when compared to brown eggs.

The third pair of ions is at 566 Da and 582 Da. The difference is 16 Da, the same as the difference between a Na+ ion and a K+ ion, for both C337 and C339. The corresponding H+ ion would be at 544 Da. In the H+ form, phosphocholine steroids fragment by loss of 183 Da. Thus, after loss of the phosphocholine, the steroid ion fragment would be at 361 Da and it would be designated as C361. MS-MS analysis did not confirm that the ion at 544 Da was the H+ cation related to the third pair (m/z= 361 +183 +1 = 544 Da). The ions with m/z= 544 Da were the Na+ ion derived from C339 (m/z= 339 + 183 + 22 = 544 Da). If the proposed structure for C361 was correct, the fragment would be an anion and would not be detected in the positive ion spectrum. C361 would have a formula mass of 378 Da when the 17 Da of the 3-hydroxy group was added. Table 3 is a trial and error analysis of the possible compositions of molecules of 378 Da and 380 Da. The only composition that leads to a molecule is C23H38O4. Panel E and F show possible structures. Panels A-H-J-K-E show a possible biosynthetic pathway that would lead from C313 to C361.

To confirm that C361 is a phosphocholine ester, the parent ion was analyzed by fragmentation. In figure 3, the parent ion was at 566 Da. At a collision voltage of 10, the molecule fragmented with loss of 59 Da. Higher voltages did not generate any additional positive ions. Loss of a fragment of 59 Da is characteristic of trimethyl amine [N(CH3)3] which would be expected if the parent molecule was a phospho-choline ester. A similar spectrum from the m/z=582 Da also showed a fragment (523 Da) characterized by loss of 59 Da from the parent. The MS fragmentation data indicates that C361 is a phosphocholine ester with a possible composition of C23H38O4. The total delta value

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must be 5. Panel E and Panel F each show one possible structure for the steroid component including a possible arrangement of the double bonds. Table 3B shows the trial and error and analysis of C363 and confirms a composition of C23H40O4, as shown in Panel G. Note that there are no alkenes present in the proposed structure.

DiscussionWhite vs Brown: The paradigm has been that the only difference between white and brown eggs is the color. The data presented here suggests there is an underlying difference between the two in the spiral steroids and, perhaps, in the electrolytes. Briefly, an egg takes

Figure 3: MSN spectrum obtained from egg white extract from white eggs. The ion collected by the trap was m/z=566 Da (C361 as the Na+ cation). The ion trap was further ionized with 10 volts to generate the spectrum shown. Further voltage did not lead to additional

observable fragmentation in the positive ion spectrum. The fragmentation demonstrated loss of 59 Da which is characteristic of trimethylamine compounds, like choline.

Line Carbons Oxygens C+0 H-Req H-Max Delta1 21 6 348 30 44 72 22 5 342 34 46 63 23 3 324 54 48 -34 23 4 340 38 48 5

5 23 5 356 22 48 136 24 3 336 42 50 47 24 4 352 26 50 128 25 3 348 30 52 11

Table 3A: Possible formulas for a molecule of mass 378 Da - C361.

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Line Carbons Oxygens C+0 H-Req H-Max Delta1 21 6 348 32 44 82 22 5 342 36 46 73 23 3 324 56 48 -44 23 4 340 40 48 4

5 23 5 356 22 48 126 24 3 336 44 50 37 24 4 352 28 50 118 25 3 348 32 52 10

Table 3B: Possible formulas for a molecule of mass 380 Da - C363. This table is a trial and error analysis of possible chemical formulas for a mass of 378 Da and 380 Da containing only carbon, oxygen and

hydrogen atoms. Each line shows a possible combination of carbon and oxygen atoms. The C+O column shows how many Da would be provided by the specific combination of carbon and oxygen atoms on that line. H-Req shows the required number of hydrogen atoms

necessary for a molecule with a mass of 378 Da. Table 3A, Line 4, (Red, italics, and underlined) shows the only combination that could make a steroid-like molecule. Note that there are actually 6 delta in the proposed structure. The 6th delta adds the 2 hydrogen atoms to make the

fragment a cation. Thus, the actual formula mass for the proposed structure of C361 is 376 Da. Table 3B, Line 4, shows the same calculation for a molecule with the proposed formula for C363.

about 24 hours to pass through the oviduct and a hen produces 5 - 7 eggs in a week. The egg is fertilized in the oviduct and then the shell is formed. After fertilization, the electrolytes in the egg whites have to be transferred to the developing chick in the yolk. Thus, there is a need for regulation of electrolyte transfer during development of the chick, but the mechanism is completely unknown.

Biosynthesis of the steroid fragment: The original paper describing the spiral steroids isolated C313, C337, C339 and C341. The conver-sion of C337 to C341 by reduction, first by 7-dehydrosterol reductase and then by 5-dehydrosterol reductase, demonstrated the last two steps in the pathway. We proposed a malonyl Coenzyme A condensation for the first step, the conversion of C313 to C337 [1]. However, the details of the process were unclear. We have now identified spiral steroid with 24 carbon atoms and one with 25 carbon atoms, C381 [2]. There are actually two reasonable structures for C381, but a malonyl CoEnzyme A condensation can’t account for the synthesis of either one. We now propose condensation with acyl-Coenzyme A derivatives as the step that adds the extra carbon atoms to C313. The steps are shown in Panels A, H, J, K, E and G. The process is equivalent to the biosynthesis of long chain fatty acids. Table 4 lists the common Coenzyme derivatives and the resulting phosphoesters that would be generated by this pathway. C359 (Panel H) would be converted to C361 by 7-dehydrosterol reductase. The carboxyl group would need to be protonated for the fragment to be detected. Thus, the observed formula for m/z=C359 Da would have a mass of 357 +17 Da (loss of the 3β-hydroxy group) or 374 Da. Earlier, we isolated two additional compounds (designated as Unknown A and Unknown B) from bovine adrenal extracts [3]. Unknown A would be designated as C361 and Unknown B would be C363. Panel E and Panel F are isomers of a fragment with m/z=361 Da, but each one has only one alkene. Thus, reduction of the alkene would produce the same product with m/z=363 Da. With the double bond, the keto-carboxyl group is planar. How-ever, if the double bond is reduced, the carboxyl group would not be in position to form the spiral lactone E-ring. This recognition suggests that neither C361 (Panel E) nor C363 (Panel G) would be a precursor or metabolite for a spiral lactone.

Biosynthesis of the phosphoester conjugate: We did not detect phosphoethanolamine steroids in either egg white or egg yolks. In bovine adrenal extracts, there was a phosphoethanolamine steroid for every phosphocholine steroid. Because there were no phospho-ethanolamine esters in either egg yolks or egg whites, the compounds in the eggs must have been synthesized elsewhere. Synthesis could

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Acyl CoA derivative R # of C Fragment Fragment Fragment Fragment

Δ5, Δ7, Δ20 Δ5, Δ20 Δ5, Δ7 Δ5 no alkeneAcetyl-CoA H 23 C337! C339! C359*! C361*! C363*!Propyl-CoA CH3 24 & C351 C353 C373* C369, C389*! &Butyl-CoA CH2CH3 25 C365! C367 C385*!

Acetoacetyl-CoA COCH3 25# C379! C381! C401*!Pentyl-CoA CH2CH2CH3 26# C379 ! C381! C401*!

Table 4: Coenzyme condensation with C313.

This is a summary of observed m/z values of phosphocholine steroid esters. Except of the ones marked with #, the trial and error analysis was consistent with only one chemical formula. We have not yet demonstrated the particular isomers, except by the pattern.

*: Compounds with carboxyl groups but the cyclic lactone has not been formed.

!: Compounds of this m/z have been detected in various species and tissues.

&: Compounds with m/z=369 and 389 Da have been detected.

Ions of these m/z values would be generated from fragments with 24 carbon atoms.

The exact isomeric structure has not yet been identified.

#: These two compounds cannot be distinguished on the basis of their observed m/z values.

Acetoacetyl-CoA is the third most common CoA derivative. The pentyl derivative has been isolated but is not widely distributed.

occur in either the ovary or the oviduct. If the egg had been fertilized, the potassium in the egg white would be needed by the developing chick in the egg yolk.

DLM: Our candidates for the endogenous cardiotonic compounds are the spiral steroid phosphocholine esters. The two current candi-dates for the endogenous DLM are ouabain [8] and marinobufagenin [9]. Both of these compounds are potent poisons and an endogenous mammalian biosynthetic pathway is not known for either one. Hamlyn isolated 11 µg of ouabain from 80 liters of human plasma [10]. No precursors for ouabagenin have been isolated from a mammalian source. A biosynthetic scheme is not known for either candidate. The glycoside portion of ouabain is rhamnose, but rhamnose is not found in mammals. Blaustein asks, why Is endogenous ouabain not ac-cepted [11] and Nicholls replies by suggesting endogenous ouabain is fantasy because (a) it cannot be detected by MS-MS analysis and (b) there is no known biosynthetic path in mammals [12]. The only method to detect ouabain in human serum is by immuno-assay. A simple experiment to demonstrate endogenous biosynthesis would be addition of radiolabeled precursor and isolation of radiolabeled ouabain. This experiment has not been reported.

Marinobufagenin is just as suspect. Marinobufagenin is extracted from toad skin with methanol [13]. The identity of the endogenous amphibian conjugate is not known. The only assay is an immunoassay [14]. The authors report the assay can detect 10 pg/ml. With an assay max of 20 times the lower limit, the upper range would be 200 pg/ml or 0.2 ng/ml. The estimated concentration of C361, C337, or C339 is 1 µg/ml or 1,000 ng/ml. Thus, if any of the spiral steroids had a cross reaction of > 0.1%, the marinobufagin assay would be detecting a spiral steroid.

This paper proposes a structure for C361 that is not an intermediate between C313 and C337. An intermediate with a carboxy group would have to have double bonds at C5-6, C7-8 and C20-23 (see Panel N) and would have an observed m/z = 357 Da and a molecular formula with 355 Da. Oysters had a C359; bovine adrenals had both C361 and C363. Thus, C363 (Panel G) had to be reduced at all three

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positions and a molecule which would generate m/z=361 Da fragment could only have 1 double bond, as shown in either Panel E or Panel F. A 3-D model of each compound shows that the proposed structure, as shown Panel E, permits free rotation of the side chain and might not align correctly for ring closure. In contrast, the Panel F structure has a rigid side chain generated by the conjugated carboxy-alkene and brings the elements of the nascent ring to close proximity. This interpretation is supported by the observation that serum from patients with 7-sterol reductase deficiency (Smith-Lemli-Opitz syndrome) have C337 but not C339 or C341. This observation indicates that the order of reduction must be Δ7 then Δ5 and the lactone ring alkene at C20-C23 must remain intact in C341 [15].

Application of immunoassay of DLM for diagnosis: If there was only one spiral steroid lactone, then, in the absence of digoxin therapy, a DLM assay might be utilized for clinical diagnosis. However, as a starting point: [a] The proportions of C337 and C339 were different between the egg whites from eggs of different colors and [b] the proportions of C339 and C341 were different between the egg yolks from eggs of different colors. Thus, as we have already noted that all four of these compounds are DLMs, a single assay will be hard to evalu-ate. Furthermore, as shown in table 4, there are three potential classes of steroid spiral lactones, each with its own steroid backbone, and there are 3 lactones in each class. These observations point to the need for specific assay methods for human diagnosis, rather than the non-specific DLM assay.

Estimated concentration: The bovine serum concentration of C341 (Ionotropin) is about 4 µM, or 2 µg/ml [1]. The relative ion intensity of C337 and C339 in the egg whites was about 20% of the intensity of miltefosine. When corrected for the dilution, the concentration was estimated at 5 µM. The only steroid with comparable concentration is DHEA-S in serum from young adults - the adult normal range is 10 - 20 µM. The fact that both DHEA-S and spiral steroid phosphoesters compounds are amphoteric, rather than lipophilic, suggests that they interact at the interface between hydrophilic and hydrophobic spaces rather than by binding to nuclear receptors.

Function: Most steroids function by binding to nuclear receptors. For example, aldosterone and corticosterone are ligands at the min-eralocorticoid receptor. When the mineralocorticoid receptor binds a ligand, protein synthesis of the epithelial sodium channel occurs, leading to sodium retention. Corticosterone is secreted episodically in response to ACTH with peak levels of 0.1 - 0.2 µM (5 - 10 µg/dl) [7]. Secretory episodes occur 5-10 times a day, but most random values are very low. Because of the episodic secretory pattern, a ligand is not usually bound to the receptor and the receptor can respond to the ligand. In contrast, although serum levels of spiral steroids do vary, there is no evidence for episodic secretion. Further, as there is no DNA in egg whites, spiral steroids couldn’t function in egg whites via a nuclear receptor. Spiral steroids are γ-lactones, a structural feature shared with spironolactone, a synthetic potassium sparing compound. Spiral steroids might function by regulating NaK-ATPase in a manner similar to spironolactone or a cardiotonic glycoside.

Conclusion• The identification of the biosynthetic pathway strengthens our theory that the spiral steroids are the long-sought DLM.

• Table 4 illustrates that there are many steroid phosphocholine esters. Isolation of individual compounds presents a tough problem. As biosynthesis of spiral steroids does not take place in eggs, there aren’t a lot of metabolites in the extracts. Thus, eggs may be a good source for isolation of the compounds in eggs.

• Egg whites have high levels of two spiral steroids, C337 and C339. This observation raises several interesting questions: {1} Is there a difference in function between the two compounds? {2} Are there drugs that function by regulation of the biosynthesis process? {3} Could this process be a target for development of novel drugs?

AcknowledgementsMarvin Applets were used for drawing, displaying and characterizing chemical structures and reactions, Product Version 19.12 Che-

mAxon (https://www. Chemaxon.com). This work was partially supported [a] by the Research Service of the United States Department of Veterans Affairs, Dr. Gary Jarvis, VA Medical Center, San Francisco, CA and Dr. Constance John, VA Medical Center, San Francisco, CA, [2] by

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a grant from the Smith-Lemli-Opitz Foundation and [3] a CRADA from IOMA to NCIRE. There was no specific funding from other agencies in the public, commercial sectors or not-for-profit sectors.

Conflict of InterestThere are no conflicts of interest.

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12. Nicholls MG., et al. “Ouabain, a circulating hormone secreted by the adrenals, is pivotal in cardiovascular disease. Fact or fantasy?” Journal of Hypertension 27.1 (2009): 3-8.

13. Fedorova O., et al. “Endogenous Ligand of α1 Sodium Pump, Marinobufagin, Is a Novel Mediator of Sodium Chloride-Dependent Hy-pertension”. Circulation 105.9 (2002): 1122-1127.

14. Abi-Ghanem D., et al. “A chemiflouresent immunoassay for the determination of marinobufagenin in body fluids”. Journal of Immunoas-say and Immunochemistry 32.1 (2011): 31-46.

15. Chasalow F and Blethen S. “On the consequences of 7-dehydrosterol reductase deficiency (SLO Syndrome)”. Presented at the Endo-crine Society Meeting (2016).

Volume 8 Issue 12 December 2019© All rights reserved by Fred Chasalow.

https://www.ncbi.nlm.nih.gov/pubmed/29550540



https://www.ecronicon.com/ecpe/pdf/ECPE-07-00267.pdf

https://www.ecronicon.com/ecpe/pdf/ECPE-07-00267.pdf

http://europepmc.org/abstract/med/12477489

http://europepmc.org/abstract/med/12477489



















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