Human Breastmilk. Perfect in Every Way. Breastmilk reveals the TRUTH about Secosteroid Hormone D.

MOTHER WARRIORS UNITE!!!!!

Water Soluble Vitamin D

NOT A MYTH

It’s the reality that matters most in the “Vitamin D” world everyone is immersed in.  I see the biggest danger being, what are we doing to the Babies?  Starting in the womb.  After you read this you will see hormone imprinting as being a much bigger deal with the entire focus being on FAT SOLUBLE D3 levels and supplementing.  And NEEDLESSLY.

This isn’t perfect but it’s a start. The beginning of the tough journey to take down the Religion of 25(OH)2D3.  A molecule which wasn’t even known to exist until nearly a decade after the first paper I’m sharing here was written about Water Soluble Vitamin D.

Think about what that means. They knew about this form of Vitamin D BEFORE the form they say is generally recognized as the serum marker for Vitamin D status. Not a storage form, they’ve never said that about the form they test, 25(OH)2D2/3.

The reality is calling it a “Vitamin” for almost a Century has made it easy to poison people with it slowly and for decades. The literature is pretty clear that Steroids are stored in a stable sulfated form. This being a “Vitamin” you could research forever and not run into sulfated BLOOD STORAGE “Vitamin D”.

Fortification.  Calling it a “Vitamin“ causes most to not even think about being fortified with it. It’s around 13 times the level naturally seen in things they spike with it like milk.

What percentage of people having conversations about D3 know the difference between a fat soluble and a water soluble vitamin?? Today’s thinking avoids the reality of storage.  True storage.  Adipose Tissue, muscle, and beyond.

“The liver is the chief storage organ, although bone, blood and kidneys also accumulate considerable amounts.”

Heftmann, E. Steroid Biochemistry. New York, NY: Academic Press; 1970. 34 p.

So we have been steered away from the form they focused on first, and in BREASTMILK, from various species.  Something that really matters.  And once they established there are copious amounts of the water soluble Vitamin D in it, they labeled it inert because it didn’t mobilize calcium. Early on this wasn’t compared directly to 25D. They had D2 and D3 but with the early science who can say they didn’t have 25D in there accidentally since they hadn’t actually “found” it yet and classified it.

So, in the 1960.s they measured the sulfated D. They didn’t compare its relative abundance to another molecule like they do today.  They knew it was abundant but called it worthless.  A mistake by the human body to be made it seems was the thinking.  “The Progression of Stupidity”, calling molecules the body made inert just because they didn’t influence calcium.

When they found 25D, around 1969/70, they thought it was the active form.

“One metabolite, 25-hydroxycholecalciferol has recently been identified and reported to act more rapidly than vitamin D3 itself. It is now believed to be the functional form of the vitamin.”

Heftmann, E. Steroid Biochemistry. New York, NY: Academic Press; 1970. 34 p.

The “Progression of Stupidity” as it relates to testing. In the beginning, they worked to make sure they found the water soluble Vitamin D, look at the early studies.  When they got that all sorted out, along came FAT SOLUBLE 25D and they stopped even looking for the Water Soluble forms all together.  But, decades later we see them being stupid trying to figure out how to test the water soluble forms again!

WTF!!

People that really care want to figure out how to test it. People that care about the science, people that care about babies, people that care about what they’re going to put the mothers through having them hormone imprint their baby, people that care about humanity in general .

As you will see, they moved their focus on 25 and 125D. And I’ve always said this is the repair pathway, you’re playing on the calcium side of the metabolism and it’s really dangerous. So, along the way they decided to up the goal in serum and they’ve thrown breastmilk under the bus along the way as well. In fact, the current position is it’s not a source of vitamin D and not only is that pathetic, it’s simply not true.  We already established packed with water soluble vitamin D.

Searching the literature, you will see that there are new tests being developed that will capture the water soluble, sulfated, forms of vitamin D. Once again it’s more abundant than its 25D counterpart and it exists every level of the metabolism that they are focused on.  There are sulfated forms of 7DHC, D3, 25D, 1,25D, and others.  And once again they’re struggling to test it, but that’s a completely different group, disconnected from the group promoting the deficiency. This would be people that care about the science.  The deficiency promoters aren’t looking at any science of true storage, this could have already been explored as it relates to liver storage of fat soluble 25D.  No one is looking to prove there isn’t a deficiency, involving storage or new forms or different pathways.  The deficiency has been embraced.  The vast majority think Vitamin D Deficiency is the cause of most human illness.  Ludicrous on its face Scientifically, especially considering is solely based upon associations with an inactive molecule as proof.

But what does that mean at the end of the day?   You and your body have FAILED. And you failed on the 10% side of the metabolism.  That’s the most insane part.  We get our lions share or D from sun.  Ten percent max orally.  Yet you failed so bad this it is now your SUPER HIGHWAY to wellness.  Not the 90% side of the metabolism.  SUN.  The “Progression of Stupidity” is strong with mainstream.    

What I see as the history here:

·         Early 60’s

·         Found it, Water Soluble D

·         Figured out testing

·         Abundant.

·         Inert

·         Forgot about it

·         Late 60’s/Early 70’s

·         Found the Vitamin D Receptors

·         Found 25(OH)2D3

·         Think it’s active.

·         Found 1,25(OH)2D3.

·         Realize it’s active, all calcium mobilization based decisions.

·         1985

·         Think they “found” sulfated 25D.  Nope. They just can tell what they have now compared to the 60’s. Nothing new

·         They RETURN TO ESTABLISHED REALITY showing water soluble D is MORE ABUNDANT than fat soluble.

·         It only took about  TWO DECADES.

Now “inertness” isn’t such a thing. They have tried to convince the world 1,25D is the answer to Cancer. To everything really. Using associations drawn with 25D levels. But they are constantly running into Hypervitaminosis D and hypercalcemia issues. Of course since no one’s measuring 1,25D the Hypervitaminosis D part stays hidden and most these folks are being documented deficient by their General Practitioners using the 25D test. Their hypercalcemia is documented but expected. They have a granulomateous disease. These balls of macrophages create a lot of 1,25D driving the hypercalcemia.

These folks running up against hypercalcemia trying to cure cancer are looking for “inert” active molecules like 1,25D. You know, like all the ones abandoned over the decades.

Today finding abundant water soluble Vitamin D in blood and breastmilk, it isn’t getting labeled “inert.” It’s getting labeled stable blood storage. Like the steroid it is. Like the other steroids. Facts. Reality.

Now I am going to turn you loose in the document.  I must warn you it is long but it is by year and I highlighted what I thought mattered most.  In some cases its most of the article.  I want to point out a few highlights about Sulfated D and then you are off.

·         Don’t impact calcium

·         Storage form

·         More abundant

·         3:1 in breastmilk

·         Sulfates at multiple levels, 7DHC, D3, 25D, 1,25D and beyond

·         Enzymes are exclusive, 25D enzymes cannot act on sulfated 25D

·         Supplements don’t increase

·         No seasonal variation

·         Sulfated forms are bound to Vitamin D Binding Protein

·         Identical affinity to VDBP as Fat Soluble forms

·         Long Stable Half-Life via VDBP

1962 (2)

Metabolic activities of vitamin D in animals.

I. Decrease of sulfate metabolism in vitamin D-deficient chicks

1. The difference of antirachitic potency between vitamin D2 and D3 was repeatedly studied using vitamin D-deficient chicks. Marked effect was confirmed in the chicks receiving vitamin D3, but it was low in the birds supplemented with vitamin D2 as judged by the chemical analyses of total ash, calcium and phosphorus.

2. The correlation between vitamin D and citrate metabolism in chicks was investigated, and the citrate and pyruvate levels in the blood and kidneys were determined and high citrate and pyruvate levels in the chicks receiving vitamin D3 was confirmed.

3. The relationship between vitamin D potency and sulfate metabolism was further studied, and the high tibia-sulfate level in the chicks receiving vitamin D3 was demonstrated using S35-sulfate. This finding suggests that there is an intimate correlation between vitamin D and sulfate metabolism in chicks.

Concerning the enzymatic role of water-soluble vitamins many investigations have been published, but few papers are available on the mechanism of the action influencing lipid-soluble vitamins. Our recent interest in the biochemistry of lipid -soluble vitamins is an outgrowth of a curiosity about the correlation between vitamin D and sulfate metabolism.

Lipmann (9) reported the mechanism of sulfate activation in chondroitin sulfate synthesis and of enzymatic transfer of "active sulfate" to steroids, galactose and hexosamine derivatives. Further, it is a well-known fact that there is a metabolic interrelation between estrogenic substances and active sulfate, as the hormones appear in female urine as the esters of glucuronic and sulfuric acids (10).

The first experiments were carried out to study the relationships between vitamin D potency and bone sulfate level. The experiments were repeated for chemical analysis of sulfate, and the data were further confirmed by injecting S35-sulfate. Thus the decrease in bone-sulfate level in vitamin D-deficient chicks was recognized by the radio-autograms of tibia. Further researches are in progress for the elucidation of the action mechanism of vitamin D in sulfate metabolism in chicks (16, 17).

5. Chemical Analysis of Sulfate in the Bone Total sulfate in the left tibia was estimated by Egami's method (19). The highest level was found in the chicks receiving vitamin D3, while the lowest in the vitamin D-deficient ones (Table V).

https://www.jstage.jst.go.jp/article/jnsv1954/8/2/8_2_121/_pdf

1963

METABOLIC ACTIVITIES OF VITAMIN D IN ANIMALS II.

ACTION OF VITAMIN D3 IN ACTIVE SULFATE METABOLISM OF CHICKS

In the foregoing paper (1) the present authors studied the relationship between vitamin D potency and sulfate metabolism and higher level of sulfate in left tibia of chicks receiving vitamin D3 was demonstrated using S35-sulfate. The researches were further repeated for the action of vitamin D3 in active sulfate metabolism of chicks. This paper deals with the outline of the results.

The conversion of inorganic phosphate to organic phosphorus compound was increased by administration of vitamin D3.

These findings seem to suggest that there may be some intimate relationship between vitamin D3 metabolism and energy metabolism relating to active sulfate (Fig. 1).

Chemical analysis of SO2-4 in left tibia were first studied. The highest level was found in the chicks receiving vitamin D3, while the lowest in the vitamin D-deficient ones (1).

https://www.jstage.jst.go.jp/article/jnsv1954/9/1/9_1_62/_pdf

1965

Metabolic activities of vitamin D in animals III.

Biogenesis of vitamin D sulfate in animal tissues

In the previous papers (1, 2), the involvement of vitamin D3 in active sulfate metabolism of chicks was reported. Further, the biogenesis of vitamin D, sulfate by rat liver homogenate from vitamin D2 was demonstrated and some physicochemical properties were studied (3, 4). In this paper, the detailed description of them will be made.

1. Formation of Vitamin D2 Sulfate by Rat Liver Homogenate Rats, weighing about 120 to 150g, were previously fed on an ordinary basal diet. After fasting for 12 hours, the animals were killed. 5g of the liver were rapidly homogenized with 7.5ml of the reaction solution containing (see paper, text not reproducible) ATP at pH 6.8 for 2 minutes. The ATP was used as a disodium salt (Tokyo Kasei Co., Ltd.). To the mixture was then added 0.5ml of propylene glycol solution of vitamin D2 containing 0.4 (see paper, text not reproducible) moles.  After incubation at 37degrees for 5 hours, the mixture was washed with ether. The etherial layer was discarded. The aqueous layer showed the formation of vitamin D2 sulfate, yielding about 0.0092 umoles.

SUMMARY

1. Enzymatic formation in vitro of vitamin D2 sulfate by various animal tissues was studied and the biosynthesis of the sulfate was proved by using the rat-liver homogenate.

2. Chemical synthesis of ammonium vitamin D2 sulfate was performed and some physicochemical properties were reported.

3. Vitamin D3 sulfate was also isolated from rabbit urine after oral administration of vitamin D3. It was in agreement with the synthesized sample.

https://www.jstage.jst.go.jp/article/jnsv1954/11/4/11_4_261/_pdf

1966

Metabolic activities of vitamin D in animals. IV.

Distribution of vitamin D sulfokinase in animal tissues and its solution

Considerable amounts of the sulfate were formed in the liver, but extremely poor in other tissues. Vitamin D-sulfokinase was isolated from rat-liver homogenate, and the optimum conditions were investigated. Isotopic investigation for the biogenesis of vitamin D sulfate was carried out using the vitamin D-sulfokinase preparation. The activity of the vitamin D2-sulfo-kinase preparation was estimated to be 3.3% as estimated from the yield of vitamin D2 sulfate from the vitamin D2 employed.

https://eurekamag.com/research/093/257/093257833.php

1966

METABOLISM OF VITAMIN D IN ANIMALS V.

ISOLATION OF VITAMIN D SULFATE FROM MAMMALIAN MILK

SUMMARY

1. The present authors have attempted to elucidate whether there may be possible occurrence of water-soluble vitamin D sulfate in fresh mammalian milk.

2. Water-soluble vitamin D sulfate was isolated as its water-insoluble barium salt from the aqueous layer of fresh deproteinized milk.

3. After repeating careful analyses, about 204 I, U. of vitamin D was shown to be present in 1 liter of fresh cow's milk in a water-soluble form, whereas about 36 I. U. per liter in a lipid-soluble form. In case of fresh human milk, it was estimated to be about 950 I. U. per liter in a water-soluble form while 15.7 I. U. in a lipid-soluble form.

https://www.jstage.jst.go.jp/article/jnsv1954/13/1/13_1_33/_pdf

1966

Metabolic activities of vitamin D in animals. VI.

Physiological activities of vitamin D sulfate

Intraperitoneal injection of mice with the sodium or ammonium salt of vitamin D2 sulfate showed it to be less toxic than free vitamin D2. The LD50 value was estimated to be 2.5 x 106 IU per kg. Three groups of rats were fed on the Steenbock diet for 8 weeks, with which one group received 10 IU vitamin D2 daily, while a second group got 10 IU of the ammonium salt of vitamin D2 sulfate. The third group acted as negative controls. It was found that vitamin D2 and its sulfate had similar potency. Doses of 5000 IU of the vitamin or its sulphated derivatives resulted after 15 days in signs of poisoning.

https://eurekamag.com/research/014/557/014557543.php

1966

Metabolic activities of vitamin D in animals: IX.

Confirmation and isolation of water-soluble vitamin D-sulfate in mammalian milks

The biogenesis of vitamin D-sulfate and isolation of vitamin D-sulfokinase in animal tissues were confirmed. The possible occurrence of water-soluble vitamin D-sulfate in fresh mammalian milks was studied. The water-soluble vitamin D-sulfate was isolated from aqueous solution of fresh protein-free milk as water-insoluble barium vitamin D-sulfate and then estimated to be about 950 International Units [I.U.] per liter of fresh human milk by thin-layer chromatography and spectrography, while as in lipid-soluble form only about 16.7 I.U. per liter. In fresh cow milk it was about 204 I.U. per liter in water-soluble form, and about 36 I.U. per liter in lipid-soluble form.

https://eurekamag.com/research/024/999/024999205.php

1966

Metabolic activities of vitamin D in animals: X.

Physiological activities of water-soluble vitamin D-sulfate

The physiological activities of the vitamin D were studied using pure synthetic vitamin D-sulfate in water-soluble form. Toxicity of vitamin D2-sulfate in saline solution was tested in mice intraperitoneally by the ordinary method. The lethal doses in mice (LD50) were estimated to be about 2,500,000 International Units [I.U.] per kilogram. The antirachitic ability of water-soluble vitamin D-sulfate was studied using the Steenbock's diet. The prophylactic dose of the water-soluble vitamin D-sulfate may be the same as that of free vitamin D.  Experiments were also carried out to determine whether hypervitaminosis appeared after administration of water-soluble vitamin D-sulfate in large doses. No toxic symptom was observed even when supplemented with 5,000 I.U. of the compound per os.

https://eurekamag.com/research/024/999/024999206.php

1967

Metabolic activity of vitamin D in animals. XI.

Antirachitic potency of the water-soluble vitamin D-sulfate in human milk

The antirachitic potency of human milk was tested by the prophylactic and curative methods using white rats which were previously fed on vitamin D-deficient diet. The animals were orally given with 2 ml of human milk (containing 780 I.U. [International Units] of vitamin D per liter). The results were shown to be positive by X-ray test and A.O.A.C.[Association of Official Agricultural Chemists] ash test.

https://eurekamag.com/research/024/999/024999261.php

1969

Antirachitic Potency of Vitamin D Sulfate in Human Milk

In the forgoing papers, the authors have reported the relation between vitamin D and active sulfate metabolism (1-4), and the isolation of vitamin D sulfate from mammalian milk (5). Further tests for the antirachitic potency of human milk (containing 780I.U. of total vitamin D in 2ml as determined by the authors) by prophyrachitic and curative methods using rats previously fed on a vitamin D - deficient diet showed the positive results both in X-ray and AOAC ash test .

Vitamin D activity of vitamin D sulfate in human milk was tested by prophylactic and curative methods using rats. The results were positive both in X-Ray and AOAC ash tests in agreement with the result of chemical assay.

The results are shown in Table 1 which suggests the presence of a considerable amount of water-soluble vitamin D in human milk.

https://www.jstage.jst.go.jp/article/jnsv1954/15/1/15_1_78/_pdf

1974

Derivatives of vitamin D3 in human and cow's milk: sulfate esters of cholecalciferol and 25-hydroxycholecalciferol

Cholecalciferol sulphate was isolated from fresh deproteinized human and cow's milk. It was characterized and estimated by thin-layer chromatography and methylene blue specific reaction. Cholecalciferol released after chemical or enzymic hydrolysis was identified and estimated by thin-layer and gas-liquid chromatography. From the unsaponifiable matter cholecalciferol and 25-hydroxycholecalciferol were isolated, separated by liquid partition chromatography on Kieselgel and Sephadex LH20 and characterized by gas-liquid chromatography; the molecular weight of 25-hydroxycholecalciferol was confirmed by mass spectrometry. Proximate constituents, estimated physico-chemically, compared with biological measurements reported in the literature indicate that the antirachitic potency of whole milk lipids is not affected significantly by the presence of traces of 25-hydroxycholecalciferol, but there is no biological evidence to suggest that the antirachitic potency is enhanced by cholecalciferol sulphate present in milk.

https://eurekamag.com/research/042/761/042761329.php

1977

VITAMIN-D IN HUMAN MILK

The vitamin-D concentration in human milk is reported to be very low, yet breast-fed infants do not develop rickets. All the earlier assays of vitamin D were made on the lipid fraction of milk, and the aqueous phase was discarded. It is now clear that most of the vitamin D in human milk is present as a water-soluble conjugate of vitamin D with sulphate.

https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(77)91764-0/fulltext

1978 (5)

Chromatographic separation of vitamin D3 sulfate and vitamin D3

Abstract

This paper describes a simple chromatographic technique on Sephadex LH20 for the separation of vitamin D3 sulfate from free vitamin D3 and its metabolites. This technique has been used in the study of vitamin D3 sulfate metabolism in rats. Seven hours after injection of vitamin D3 sulfate (35S or 35S and 3H) only the peak of vitamin D sulfoconjugate was found in chromatographic elution of serum extracts.

https://pubmed.ncbi.nlm.nih.gov/209579/

1979 (1)

Vitamin D3 sulfoconjugate distribution and evolution in pregnant or lactating rats

Abstract

24 hrs after intravenous injection of vitamin D3 35S sulfoconjugate the radioactivity in kidney is about twice as much in pregnant Rats as in lactating Rats. It decreases from 24 to 48 hrs in pregnant but increases in lactating Rats kidneys. Hydrolysis of vitamin 3H D3 sulfate is detected in kidney and liver extracts, the ratio of free vitamin D3 on vitamin D3 sulfate is higher in pregnant than in lactating animals. Mammary glands of lactating Rats contain mainly the unchanged vitamin D3 sulfate which can be hydrolysed by new born suckling pups.

https://pubmed.ncbi.nlm.nih.gov/221128/

1980

Lack of biological activity of vitamin D3-3 beta sulfate during lactation in vitamin D-deficient rats

Abstract

Sulfoconjugated vitamin D has been claimed to have an important antirachitic activity and to be present at higher amounts than free vitamin D in maternal milk. We have previously shown that vitamin D3-3 beta sulfate (SD3) administration has little effect on calcium and bone metabolism during pregnancy in rats. In the present work, we have compared the biological activity of free vitamin D3 (D3) and SD3 during the lactation period. After delivery, D-depleted (-D) female rats were orally treated with D3 or SD3 (1,300 pmoles/every two days) during 20 days of lactation. Vitamin D status was determined before, during and at the end of the treatment for mother rats and at days 1 and 20 of life in suckling pups. Both mothers and pups were sacrificed at day 20 of lactation and subjected to hormonal and mineral determinations and to histomorphometric analysis of bone metabolism. After 12 days of SD3 treatment, mother rats showed a slight but significant elevation in plasma concentrations of calcium phosphorus and vitamin D metabolites. This effect was reversed at the end of lactation; at this time most maternal plasma parameters did not differ from those observed in -D non-treated mothers. By contrast, 20 days of D3 administration in mothers normalized plasma biochemical parameters. These results were confirmed by analysis of both static and dynamic parameters of bone formation. Maternal SD3 treatment did not improve either plasma biochemical or histological parameters of bone formation and resorption in suckling pups which remained comparable to that of D-deficient pups; by contrast, pups from D3-treated mothers normalized most biochemical plasma parameters although bone metabolism remained abnormal. In conclusion, the biological activity of SD3 on bone and mineral metabolism during lactation in rats is as low as in the nonreproductive stages.

https://pubmed.ncbi.nlm.nih.gov/2834806/

1981 (1)

Synthesis and Biological Activity of Vitamin D3-Sulfate*

Vitamin D­3-3Bsulfate has been synthesized using pyridine sulfur trioxide as the sulfate donor. It has been shown to be pure by high performance liquid chromatography and spectral methods. Unlike previous reports, the product has been identified unambiguously as the 3B-sulfate ester of vitamin D3 by its ultraviolet, nuclear magnetic resonance, infrared, and mass spectra. The biological activity of vitamin D3-sulfate was then determined in vitamin D-deficient rats. Vitamin D3-sulfate has less than 5% of the activity of vitamin D3 to mobilize calcium from bone and approximately 1% of the ability of vitamin D3 to stimulate calcium transport, elevate serum phosphorus, or support bone calcification. These results disprove previous claims that vitamin D3-sulfate has potent biological activity, and they further do not support the contention that vitamin D-sulfate represents a potent water-soluble form of vitamin D in milk.

In recent years it has been suggested that conjugation of vitamin D with sulfate (so,) (Fig. 1) plays an important role in vitamin D metabolism. Higaki et al. (1) reported the in vitro formation of vitamin D2-SO4 by rat liver homogenates. They further suggested that vitamin D2-SO4 has approximately the same antirachitic potency as vitamin D2 itself (2) but is less toxic when administered in large amounts (3). Unfortunately the authors failed to provide convincing evidence that the vitamin D2-SO4 had been prepared nor was satisfactory evidence of purity offered, casting doubt on the reported biological activity. Miravet et al. (4) repeated the experiments of Higaki using vitamin D3, but they also failed to provide evidence of purity or physical evidence that the correct product was obtained. Thus, the reported potent biological activity of the vitamin D3-3b-sulfate remained in considerable question. The sulfate ester of vitamin D has also been isolated from the urine of both rabbits (1) and rats (5) after oral administration of massive amounts of the vitamin. High levels of a water-soluble form of vitamin D activity have been reported to occur in milk. Sahashi et al. (6) found cows’ milk to contain 240 IU of vitamin D activity per liter, of which 80% was claimed to be water soluble. In addition, these authors report that human milk contains 965 IU of vitamin D activity per liter. Similar findings have been reported by Lakdawala and Widdowson (7). Both studies infer that this water-soluble form of vitamin D is the sulfate conjugate. Since it is generally believed that even fairly minor structural changes greatly affect the biological activity of analogs of vitamin D (8), it was of great importance to determine if vitamin D3-S04 has biological activity and if it acts upon all of the target organs of vitamin D. The results of this study demonstrate that vitamin D3-S04 has little or no biological activity, in contrast to previous reports.

Vitamin D3-S04 exhibits very low activity in all of the classical assays for the functions of vitamin D. The data in Table I demonstrate that vitamin D3-S04 retains less than 1% of the ability of vitamin D3 to stimulate duodenal calcium transport. No stimulation of calcium mobilization from bone could be detected even when 6.5 nmol/day of vitamin D3-S04 was administered (Table II). Vitamin D3-S04 elevated the serum phosphorus concentration of phosphorus-depleted, vitamin D-deficient rats only after 6.5 nmol/day of the compound was administered (Table III). Approximately 1% of that amount of vitamin D itself causes a similar response. Calcification of newly formed bone could be detected only after 6.5 nmol/day of vitamin D3-S04 was administered orally.

DISCUSSION

The biological activity of vitamin D3 is reduced to less than 5% by conjugation of the 3P-hydroxyl with sulfate. This very low biological activity indicates that vitamin D3-S04 has little or no activity itself. In addition, the sulfate ester is evidently stable under physiological conditions, and very little vitamin D is released in vivo from the substrate. This might be expected, since at room temperature vitamin D-SO4, is stable in base and is only slowly hydrolized to release vitamin D in acid (2). It is likely that vitamin D3-S04 is hydrolyzed slowly in the stomach when the compound is administered orally. This would account for the slightly higher biological activity observed under these conditions. The present results are in direct contrast to reports of high biological activity of vitamin D2-S04 (3, 13) and vitamin D3- SO4 (4). In neither of those cases did the authors provide adequate evidence that the vitamin D-sulfate was obtained nor did they provide convincing evidence that the product was sufficiently pure for meaningful biological evaluation. As a result of those and other (1-7) similar reports there has been the widespread belief among nutritionists that vitamin D sulfate has potent biological activity. The present study clearly dispels this belief. The results of the present study also do not support the idea that vitamin D-sulfate is a potent form of vitamin D in milk (6, 7); if the sulfate-ester is in fact present in milk, it has little or no biological activity. Furthermore, the methods used in previous studies to assess the presence of vitamin D-sulfate in milk are questionable, making the results difficult to evaluate. This question must, therefore, be re-examined. The question of whether vitamin D-sulfate is an important excretory form of the vitamin has not been addressed in the present study. The significance of earlier reports of vitamin D3-S04 as an excretory product in animals (1,4) is difficult to assess, in view of the massive amounts of vitamin D3 administered in these studies and the very low abundance of sulfate ester recovered. This question should be examined more closely before its role as an excretory product can be assessed. At present, however, the concept of a highly biologically active vitamin D-sulfate can be discarded.

https://www.jbc.org/article/S0021-9258(19)70051-9/pdf

1981 (2)

Occurrence of Vitamin D Sulfate in Human Milk Whey

Following reports that vitamin D sulfate is the major source of vitamin D activity in human milk, we investigated the presence of this compound in milk whey using a modification of techniques for the determination of vitamin D metabolites in plasma. Synthetic cholecalciferol sulfate, ergocalciferol sulfate and [3H]cholecalciferol sulfate were prepared by reacting radioactive cholecalciferol or nonradioactive cholecalciferol or ergocalciferol with sulfamic acid in pyridine. The products were purified sequentially by Sephadex LH-20 and high pressure liquid chromatography. The purified products were chromatographically homogenous, exhibited an ultraviolet absorption spectrum identical to that of standard cholecalciferol, demonstrated a sulfonate ester linkage and upon saponification yielded the parent vitamin. Milk whey was extracted with methanol:methylene chloride (1:2, v/v) using [3H]cholecalciferol sulfate to estimate recovery of the compound. The extract was purified by chromatography on silica cartridges and reverse phase high pressure liquid chromatography and was quantitated by ultraviolet absorption (UV). Although added cholecalciferol sulfate was readily detected in human milk whey samples, no endogenous vitamin D sulfate was found (detection limit 1 ng/ml). The results indicate that vitamin D sulfate is not a major source of vitamin D activity in human milk.

1981 (6)

Synthesis and biological activity of vitamin D3 3 beta-sulfate. Role of vitamin D3 sulfates in calcium homeostasis

To determine the biological activity of vitamin D sulfates, we synthesized vitamin D3 3P-sulfate and tested its biological activity in vitamin D-deficient hypocalcemic rats. When vitamin D3 sulfate was administered as a single oral dose of 208,000 or 416,000 pmol (100 pg or 200 pg), it increased active calcium transport in the duodenum and was also able to mobilize calcium from bone and soft tissue. Dose levels below this failed to elicit a response. Vitamin D3 itself was active at doses as low as 260 pmol when administered in this manner. In order to test the biological activity of vitamin D3 sulfate in various doses when administered chronically, we tested the biological activity of vitamin D3 sulfate after 5 days of oral dosing: vitamin D3 sulfate was active at doses of 52,000 pmol/day (25 pg), whereas vitamin D3 was active at doses of 65 to 260 pmol/day over a period of 5 days. When administered as a single intravenous dose, vitamin D3 sulfate exhibited no biological activity in doses as high as 52,000 pmol. Vitamin D3, however, was active at a dose of as low as 65 pmol. We conclude that vitamin D3 sulfate, a metabolite of vitamin D3 of heretofore unknown biological activity, is considerably less active than vitamin D3 itself.

…it has been postulated that because breast-fed infants do not become rachitic, the antirachitic activity of milk is probably due to the biological activity of vitamin D sulfates. It has been hypothesized that the hydrolysis of vitamin D sulfate to free vitamin D could result in the formation of sufficient vitamin D to prevent rickets. Various workers have claimed that human milk contains between 1 to 2 pg/dl of putative vitamin D sulfate (Lakdawala and Widdowson, 1977).

In any event, our results using biological tests of vitamin D3 function lead us to believe that young rats can utilize vitamin D3 sulfate when it is administered chronically via the oral route at doses of 52,000 pmol or higher. Whether vitamin D3 sulfate acts without being hydrolyzed to vitamin D3 or acts after hydrolysis to free D3 is not very clear. We conclude that the vitamin Ds sulfate is biologically active only in high doses when administered orally to young rats. It is possible that vitamin D sulfate could be utilized by infants as a source of vitamin D. Whether or not the amounts present in milk can be utilized efficiently by the human infant remains unclear.

https://pubmed.ncbi.nlm.nih.gov/6263879/

1982 (1)

The Vitamin D Activity of Milk

Recent studies raise doubts that human milk and cow's milk contain vitamin D sulfate or that the known levels of vitamin D and its metabolites in milk are sufficient to meet the requirements of infants.

…neither human nor cow’s milk has significant anthracitic activity for rats.

https://eurekamag.com/research/001/020/001020514.php

1982 (2)

Water-Soluble Vitamin D in Human Milk: A Myth

For the past 50 years, repeated analyses of human milk have shown an average of 20 IU/liter of vitamin D activity. Following the 1977 report of Lakdawala and Widdowson,1 which found that a large amount of water-soluble vitamin D sulfate was present in the whey (or water-soluble) fraction of human milk, the previous studies appeared to be in question. This study by Lakdawala and Widdowson reportedly confirmed earlier reports from Japan2-4 and France5 which not only demonstrated large quantities of vitamin D sulfate in human milk, but also strongly suggested that this vitamin D metabolite had biologic activity in rats.

https://publications.aap.org/pediatrics/article-abstract/69/2/238/50850/Water-Soluble-Vitamin-D-in-Human-Milk-A-Myth

1982 (4)

Cholecalciferol sulfate identification in human milk by HPLC

Abstract

Synthetic vitamin D3 sulfate was prepared by reacting cholecalciferol with sulfamic acid in pyridine. Vitamin D3 sulfate ammonium salt was purified by crystallisation and transformed in sulfate sodium salt. Homogeneity was controlled by reverse phase high pressure liquid chromatography (HPLC). Purified synthetic vitamin D3 sulfate sodium salt was used as a reference. Milk whey was obtained after protein precipitation by adding ethanol. Vitamin D3 sulfoconjugate was identified in supernatant (lyophylized) after purification by Sephadex LH 20 and HPLC. Milk whey purified fraction obtained exhibited the same ultra-violet absorption (UV) as synthetic vitamin D3 sulfate; after solvolysis, cholecalciferol was liberated from natural and synthetic sulfoconjugate. The results confirmed that vitamin D3 sulfate was present in human milk.

https://www.sciencedirect.com/science/article/abs/pii/0039128X82900630?via%3Dihub

1982 (11)

Synthesis and biological activity of vitamin D2 3β-glucosiduronate and vitamin D2 3β-sulfate: Role of vitamin D2 conjugates in calcium homeostasis

Abstract

To ascertain the physiologic function of vitamin D2 conjugates in calcium homeostasis, we synthesized vitamin D2 3β-glucosiduronate and vitamin D2 3β-sulfate in pure form and tested their biological activity in vitamin D deficient rats fed a low calcium diet. Vitamin D23β-glucosiduronate was active in promoting calcium transport in the intestine at a dose of 100 pmol per rat. It increased calcium mobilization from bone and soft tissue at a dose of 1000 pmol per rat. This conjugate was less active than equimolar doses of vitamin D2. These results demonstrate that vitamin D2 3β-glucosiduronate can be utilized by the rat as a source of vitamin D. In contrast, vitamin D23β-sulfate was biologically inert. It failed to increase calcium transport in the duodenum of vitamin D deficient rats except at the highest doses tested (≫100,000 pmol/rat). It was similarly ineffective in increasing calcium mobilization from bone and soft tissue. Our results lead us to conclude that vitamin D23β-glucosiduronate is probably utilized by the rat after hydrolysis to the free sterol; on the contrary, the sulfate is not biologically active except at the highest doses tested.

https://mayoclinic.elsevierpure.com/en/publications/synthesis-and-biological-activity-of-vitamin-dsub2sub-3β-glucosid

1985

Vitamin D3 3 beta sulfate has less biological activity than free vitamin D3 during pregnancy in rats

The biological activities of free (D3) and sulfoconjugated (SD3) vitamin D3 were compared after 6 weeks of oral administration to D-deficient (-D) female rats which were mated in the meantime. Mothers and pups were sacrificed 1-2 days following parturition and mineral and hormonal plasma status was determined in mothers and bone mineral determinations and bone histomorphometric studies performed. In newborns, plasma levels of Ca, P and 25-hydroxyvitamin D (25(OH)D) were measured. After parturition, -D mothers had decreased body weight (BW) as well as decreased plasma levels of Ca, P and 1,25-dihydroxyvitamin D (1,25(OH)2D) associated with undetectable levels of 25(OH)D. Plasma levels of immunoreactive calcitonin and parathormone, by contrast, were higher than in vitamin D-replete (+D) control mothers. Bone histomorphometric analysis showed osteomalacia and secondary hyperparathyroidism in -D mothers. After parturition, -D +SD mothers had reduced BW compared to D-treated mothers and the plasma parameters measured were abnormal. Almost all bone histomorphometric parameters were found to be intermediate between +D and -D groups without reaching values of +D mothers. By contrast, -D +D mothers had most of the bone formation parameters identical to those of +D mothers. However, bone resorption was still higher while plasma levels of P and 25(OH)D remained slightly, but significantly lower than in +D mothers. In pups, plasma Ca in both D3- and SD3-treated groups was similar to values in +D-treated rats. However, pups from SD3-treated mothers still showed plasma levels of P and 25(OH)D lower than in +D pups. In conclusion, treatment with SD3 in -D mother rats significantly improves the biochemical plasma parameters of pups, but complete normalization can be achieved only in the D3-treated group. Our results show that when administered at equal amounts, SD3 has a much lower biological activity than D3 in -D female rats and cannot therefore replace vitamin D3 particularly during pregnancy.

https://pubmed.ncbi.nlm.nih.gov/2998494/

1985 (10)

25-Hydroxyvitamin D3 3-sulphate is a major circulating form of vitamin D in man

The mean concentration of sulphated 25-hydroxyvitamin D, in plasma from 60 patients was 16.7+/- 7.1 ng/ml and the levels often exceeded those of the corresponding free compound. The study also shows that unconjugated 25-hydroxvitamin D, is not readily sulphated by man in vivo.

Here I report that 25-hydroxyvitamin D3 3b-sulphate is a major circulating form of vitamin D3 in man. The concentration of the sulphate often exceeds that of free 25-hydroxyvitamin D3, previously recognized as the quantitatively major metabolite in blood [l-4].

3.2. Concentration in human plasma

The concentrations of 25hydroxyvitamin D3 3-sulphate and of the corresponding free compound in plasma from 10 apparently healthy human subjects were determined by HPLC (fig.3).

Addition of 3H-labelled free reference compound following separation of conjugates served to correct for degradation and losses during solvolysis and further purification. No correction for losses prior to solvolysis was made, however, yields of the 3H-labelled sulphate added to plasma were essentially quantitative. During winter (February) the mean concentration of sulphated 25-hydroxyvitamin D3 in plasma was 21.1 +/- 9.2 ng/ml, which was similar to that of the corresponding free compound (mean 19.8 +/- 10.1 ng/ml). During summer (August) the concentration of the free compound was increased in 8 subjects (mean 29.9 +/- 9.9 ng/ml), and exceeded the sulphate levels in all subjects studied (mean 21.9 +/- 7.2 ng/ml). Thus, the seasonal variation of plasma levels of free 25-hydroxyvitamin D3 previously demonstrated [19] and also seen here was not observed for the sulphated compound, indicating that its formation does not directly reflect the levels of the free compound. However, statistical evaluation of levels of the free and sulphated compound in plasma from 60 patients (mean 17.1 +/- 7.2 ng/ml and 16.7 +/- 7.0 ng/ml, respectively) provided strong evidence for a relationship between the 2 compounds (fig.3). Great differences in concentrations of the 2 compounds have been observed in plasma from a few subjects, e.g. the levels of the free and sulphated 25-hydroxyvitamin D3 in plasma from a 4-month-old child (given vitamin D2 as supplement) were 0.7 and 12.6 ng/ml, respectively. The significance of this result is not yet understood.

A dietary source of the compound or its precursor cannot be excluded, and vitamin D3 sulphate has previously been detected in significant amounts in dairy milk [5,6].

Vitamin D3 sulphate has also been found in milk from lactating women [5-8] but its proposed role in preventing rickets in breast-fed infants has been questioned [23,24]. Recent studies have shown that vitamin D3 (and D2) sulphate possesses little or no biological activity [25-27]. In an early study, 25-hydroxyvitamin D3 sulphate was detected as a minor component in milk from women [6]. When reanalyzed by the same procedure as described here for plasma, levels in milk collected from 4 women a few days after delivery were much lower than in plasma and below the detection limit (<I ng/ml) in all samples. Therefore, it does not seem likely that 25hydroxyvitamin D3 sulphate is the ‘antirachitic agent’ in human milk.

This study has shown that the sulphate of 25-hydroxyvitamin D3 is a quantitatively important form of secosteroids in man. Whether it possesses biological activity, reflects the presence of a new biosynthetic pathway to hydroxylated vitamin D3, or is an inactive excretion product remains to be established.

https://febs.onlinelibrary.wiley.com/doi/abs/10.1016/0014-5793%2885%2980002-8

1986 (2)

Assessment of vitamin D sulphate in human milk using desorption chemical ionization mass spectrometry

Vitamin D3 sulphate (SD3) identification in human milk was obtained using Desorption Chemical Ionization (DCI). The chemical ionization reagent gas used was nitrogen, molecules were ionized when the emitter was heated. SD3 was obtained from lactarium human milk and purified by high-performance liquid chromatography (HPLC). A selected ion monitoring (SIM) measurement was carried out with typical ions, m/z 366 for SD3 and m/z 384 for parent vitamin D3, the intensity ratio (I366/I384) greater than 1 being related to the presence of the sulphoconjugated form of vitamin D3 in the sample analysed. The detection of small quantities of SD3 in human milk is possible using this technique.

https://pubmed.ncbi.nlm.nih.gov/3006846/

1987 (3)

The cholecalciferol sulphate system in mammals

Abstract

7-Dehydrocholesterol sulphate has been identified in human and rat skin. The compound was isolated by anion exchange chromatography and following hydrolysis it was characterized by high-performance liquid chromatography and gas chromatography-mass spectrometry. Experiments with rats showed that 7-dehydrocholesterol sulphate can serve as a precursor of cholecalciferol sulphate and 25-hydroxy-cholecalciferol 3-sulphate, the latter compound being present in significant amounts in human blood. The sulphated sterols identified represent a previously unknown secosteroid system in mammals.

https://pubmed.ncbi.nlm.nih.gov/3035283/

1988 (7)

Vitamin D metabolism in human pregnancy. Concentrations of free and sulphated 25-hydroxyvitamin D3 in maternal and fetal plasma at term

“The concentrations of free and sulphated 25-hydroxyvitamin D3 in 20 paired maternal-cord plasma samples obtained at delivery have been determined. The compounds were isolated by liquid-solid extraction at elevated temperature, and the sulphate was purified by anion exchange chromatography prior to hydrolysis and analysis by high-performance liquid chromatography. The study shows that unconjugated 25-hydroxyvitamin D3 is predominant in maternal plasma (mean 20 ng/ml) whereas the sulphate is the major form of vitamin D3 in fetal circulation (mean 21 ng/ml plasma). The total concentration of the two compounds in cord plasma (mean 35 ng/ml) was significantly higher than that in maternal plasma (mean 30 ng/ml). Positive correlations were obtained between maternal and cord plasma levels of free 25-hydroxyvitamin D3, between maternal and cord plasma levels of sulphated 25-hydroxyvitamin D3 and between plasma levels of the maternal free compound and the fetal sulphate. There was also a relationship between the levels of free and sulphated 25-hydroxyvitamin D3 in cord plasma. The results suggest that sulphation may be a physiologically important reaction for deactivating and/or trapping secosteroids in the fetus.”

https://www.sciencedirect.com/science/article/abs/pii/0022473188902026

1988 (8)

Improvement of dental development in osteopetrotic mice by maternal vitamin D3 sulfate administration

Abstract

Utilizing the microphthalamic mouse, (mi/mi) as a model of osteopetrosis, vitamin D3 (cholecalciferol) was administered prenatally and postnatally to study its effects on tooth development and subsequent eruption. It has previously been reported that vitamin D3 crosses the placental barrier and is absorbed into mammary gland milk. Fifteen heterozygotes (+/mi) were used as breeders. There were three study groups: A) 5.0 ng/gm cholecalciferol sulfate; B) 2.5 ng/gm cholecalciferol sulfate; and C) no therapy. Intraperitoneal injections were administered three times per week, beginning when pregnancy was evident, and continuing for 4 additional weeks during lactation. Approximately half of the 59 offspring were sacrificed at age 1 day and the other half at 4 weeks. The former group was studied for crown development, and the latter group was studied for root development and eruption. When the osteopetrotic offspring of group A were compared with osteopetrotic offspring of group C, crown development and tooth eruption were substantially more advanced. Parameters examined were maturity of the ameloblasts and odontoblasts, dentin and enamel formation, root sheath development, status of eruption, and degree of apex closure. It was concluded that cholecalciferol sulfate significantly improves tooth development and subsequent eruption in the osteopetrotic mouse. A genetic disease has had its phenotype modified by vitamin therapy during gestation.

https://pubmed.ncbi.nlm.nih.gov/2850297/

1989 (3)

Modulation of cell—cell and cell—antigen interactions by 1,25-dihydroxyvitamin D3 and vitamin D3 sulfate in vitro: a study on pregnancy lymphocytes and hybridoma cells

The effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) was investigated in single cell cytotoxicity assays, using K-562 target cells. The action of vitamin D3 sulfate (VD3S) in natural cytotoxicity assays as well as its effect on the antigen-specific adherence of hybridoma cells has also been studied. In the single cell cytotoxicity assay 1,25(OH)2D3 dose-dependently and significantly increased the binding of PBMC to target, the number of lysed target cells and NK activity. RU486, a compound known as a potent blocker of progesterone and glucocorticoid receptors, suppressed the effect of 1,25(OH)2D3 in all systems. VD3S dose-dependently decreased the natural cytotoxicity of PBMC and the binding of hybridoma cells to antigen immobilized on plastic surfaces. The results suggest that both 1,25(OH)2D3 and VD3S are potent modulatory agents in cell-cell and cell-antigen interactions.

https://pubmed.ncbi.nlm.nih.gov/2541082/

1991 (3)

Human placental sterylsulfatase. Interaction of the isolated enzyme with substrates, products, transition-state analogues, amino-acid modifiers and anion transport inhibitors

Abstract

The enzymatic properties of a homogeneous sterylsulfatase preparation isolated from human term placenta were studied. The enzyme exhibited both arylsulfatase and sterylsulfatase activity: it catalysed the hydrolysis of sulfuric acid esters of (in the order of decreasing specific activity) non-steroidal phenols, of a phenolic steroid, and of neutral 3 beta-, 21- and (though at a very low rate) 17 beta-hydroxysteroids. However, among all the substrates tested only the 3-sulfates of phenolic and neutral steroids exhibited high affinity towards the sulfatase. Vitamin D3 sulfate was not hydrolysed by the sterylsulfatase but strongly inhibited its activity. The products of the catalytic reaction, free steroids or phenols as well as the sulfate anion or analogues thereof, likewise interfered with the enzyme's activity. Ki values of unconjugated steroids were ten- to hundredfold higher than Km values of the respective sulfoconjugates. Inorganic sulfate only slightly inhibited the sulfatase activity; its inhibitory potency, however, increased in a time-dependent manner when it was preincubated with the enzyme prior to assay. In contrast to sulfate, the hypothetical transition-state analogues sulfite and vanadate acted as strong inhibitors of the sulfatase activity. According to the results of an analysis of the effect of pH on sterylsulfatase kinetics, enzyme constituents with pK values of approximately 5.8 and 8.0 are involved in a general acid-base catalysed reaction. Treatment of the sulfatase with amino-acid side chain modifying reagents directed against arginine, cysteine, cystine, serine or tyrosine residues did not result in significant alteration of its activity. Diethyl-pyrocarbonate known to react primarily with histidyl groups, however, rapidly inactivated the enzyme; this inactivation reaction was markedly retarded in the presence of substrate. Histidine thus appears to be essential for the catalytic activity of the sulfatase. Taken together, the present results reveal a considerable similarity between the catalytic mechanism of human placental sterylsulfatase and the ones already proposed for the lysosomal arylsulfatases A and B. Taurocholate, salicylate, ouabain, and 4,4'-substituted stilbene-2,2'-disulfonates are well known inhibitors of carrier-mediated transport of anions across cellular membranes. With the exception of ouabain, these compounds likewise turned out to inhibit the enzymatic hydrolysis of steryl sulfates; the pattern of dose dependences of their interference with the sulfatase activity resembles the one reported for inhibition of anion transport. Since the sterylsulfatase in vivo strongly is associated with cellular membranes including the plasma membrane of the syncytiotrophoblast, this finding supports the speculation that similar molecular structures may be involved in both placental transport and hydrolysis of anionic steryl sulfates.

https://pubmed.ncbi.nlm.nih.gov/1828947/

1994 (4)

Lack of inhibition of placental estrone sulfatase and aromatase enzymes by vitamin D3 and its analogs

Abstract

The aromatase and estrone sulfatase enzymes are important sources of biologically active estrogens in postmenopausal women with breast cancer. Promising initial results in the treatment of endocrine-responsive breast cancer have been exhibited by 1 alpha 25-dihydroxyvitamin D3 and the synthetic vitamin D analogues MC903 and EB1089. However, these compounds together with vitamin D3 and vitamin D3 sulfate did not inhibit the human placental aromatase enzyme when assayed up to 20 microns. Only vitamin D3 sulfate and 1 alpha 25-dihydroxyvitamin D inhibited the estrone sulfatase activity in human placental microsomes, albeit at high concentration (32 and 37% inhibition, respectively with 50 microns each inhibitor). It is unlikely that inhibition of aromatase or estrone sulfatase enzymes contribute to the inhibitory effect of this group of compounds on breast cancer.

https://pubmed.ncbi.nlm.nih.gov/8180120/

1995 (2)

Separation and characterization of 25-hydroxyvitamin D3 3-sulfate in human plasma by high-performance liquid chromatography

The separation and characterization of 25-hydroxyvitamin D3 3-sulfate in human plasma are carried out by high-performance liquid chromatography. The vitamin D sulfate fraction is obtained from a plasma specimen (three volunteers) by the combined use of a Sep-Pak C18 cartridge, for solid-phase extraction, and a lipophilic gel (piperidinohydroxypropyl Sephadex LH-20), for ion-exchange chromatography. 25-Hydroxyvitamin D3 3-sulfate is identified in the examined three specimens in two ways: its chromatographic behavior and that of its fluorescent labeled derivative using 4-[4-(6-methoxy-2-benzoxazolyl)phenyl]-1,2,4-triazoline-3,5- dione; and data obtained from the solvolysis reaction.

https://pubmed.ncbi.nlm.nih.gov/7876374/

1995 (9)

Quantitative determination of 25-hydroxyvitamin D3 3-sulphate in human plasma using high performance liquid chromatography

The quantitative determination of 25-hydroxyvitamin D3 3-sulphate in human plasma was completed using reversed phase high performance liquid chromatography with UV detection (265 nm) and an internal standard method. The vitamin D sulphate fraction was obtained from a plasma specimen with the combined use of a Bond Elut C18 cartridge for solid-phase extraction and a piperidinohydroxypropyl Sephadex LH-20 column for lipophilic ion-exchange chromatography. Separation of the compounds was performed on a YMC-Pack ODS-AM column. The limit of quantitation was 5 ng/mL and the assay was linear from 5 to 50 ng/mL. The proposed method is satisfactory in its accuracy and precision.

https://pubmed.ncbi.nlm.nih.gov/8593424/

1999 (1)

Levels of 24,25-dihydroxyvitamin D3, 25-hydroxyvitamin D3 and 25-hydroxyvitamin D3 3-sulphate in human plasma

Abstract

The concentrations of (24R)-24,25-dihydroxyvitamin D3[24,25(OH)2D3], 25-hydroxyvitamin D3[25(OH)D3] and its 3-sulphate [25(OH)D3(3)S] in the plasma of healthy subjects, patients with chronic renal failure, patients with climacteric syndrome, pregnant women and foetuses were determined using the enzyme-linked immunosorbent assay and high-performance liquid chromatography. 25(OH)D3(3)S was not detected in about one-third of the plasma samples from patients with chronic renal failure (n = 26). The three metabolites in maternal plasma reached the highest levels in the second trimester of pregnancy followed by a decrease to the values obtained in the first trimester. Older healthy women (age range 44-71 years) showed higher levels of 24,25(OH)2D3 and 25(OH)D3 in the plasma than did young healthy women (age range 21-29 years), whereas no clear difference was observed between the older healthy women and patients with climacteric syndrome. The level of 25(OH)D3(3)S in the plasma was higher in the latter patients than in healthy women.

https://pubmed.ncbi.nlm.nih.gov/10370759/

2004 (10)

Critical role of vitamin D in sulfate homeostasis: regulation of the sodium-sulfate cotransporter by 1,25-dihydroxyvitamin D3

Abstract

As the fourth most abundant anion in the body, sulfate plays an essential role in numerous physiological processes. One key protein involved in transcellular transport of sulfate is the sodium-sulfate cotransporter NaSi-1, and previous studies suggest that vitamin D modulates sulfate homeostasis by regulating NaSi-1 expression. In the present study, we found that, in mice lacking the vitamin D receptor (VDR), NaSi-1 expression in the kidney was reduced by 72% but intestinal NaSi-1 levels remained unchanged. In connection with these findings, urinary sulfate excretion was increased by 42% whereas serum sulfate concentration was reduced by 50% in VDR knockout mice. Moreover, levels of hepatic glutathione and skeletal sulfated proteoglycans were also reduced by 18 and 45%, respectively, in the mutant mice. Similar results were observed in VDR knockout mice after their blood ionized calcium levels and rachitic bone phenotype were normalized by dietary means, indicating that vitamin D regulation of NaSi-1 expression and sulfate metabolism is independent of its role in calcium metabolism. Treatment of wild-type mice with 1,25-dihydroxyvitamin D3 or vitamin D analog markedly stimulated renal NaSi-1 mRNA expression. These data provide strong in vivo evidence that vitamin D plays a critical role in sulfate homeostasis. However, the observation that serum sulfate and skeletal proteoglycan levels in normocalcemic VDR knockout mice remained low in the absence of rickets and osteomalacia suggests that the contribution of sulfate deficiency to development of rickets and osteomalacia is minimal.

https://journals.physiology.org/doi/full/10.1152/ajpendo.00151.2004

2014 (10)

Development and validation of a method for determination of plasma 25-hydroxyvitamin D3 3-sulfate using liquid chromatography/tandem mass spectrometry

25-Hydroxyvitamin D3 3-sulfate [25(OH)D3S], the sulfated conjugate of 25(OH)D3, is reported to be another major metabolite of vitamin D3, and its circulating level is similar to or higher than that of 25(OH)D3 in adults or infants, respectively [5], [11], [12], [13], [14]. Although the biological role of 25(OH)D3S is not still fully understood, it might be the storage form of vitamin D3. Therefore, it is expected that the quantification of 25(OH)D3S in plasma/serum is also helpful in the assessment of the vitamin D status, especially for infants. A method using high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection has been reported for the quantification of plasma 25(OH)D3S [13]; the method, however, employed a relatively-large volume of sample (0.5 mL) and complicated pretreatment procedures (2 steps of solid-phase extraction and ion-exchange chromatographic purification after deproteinization), and is not necessarily suited to evaluate the diagnostic value and biological role of 25(OH)D3S.

https://www.sciencedirect.com/science/article/abs/pii/S1570023214005443

2015 (12)

Determination of four sulfated vitamin D compounds in human biological fluids by liquid chromatography-tandem mass spectrometry

Abstract

The determination of both the water-soluble and lipid-soluble vitamin D compounds in human biological fluids is necessary to illuminate potentially significant biochemical mechanisms. The lack of analytical methods to quantify the water-soluble forms precludes studies on their role and biological functions; currently available liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods are able to determine only a single sulfated form of Vitamin D. We describe here a highly sensitive and specific LC-MS/MS method for the quantification of four sulfated forms of vitamin D: vitamins D2- and D3-sulfate (D2-S and D3-S) and 25-hydroxyvitamin D2- and D3-sulfate (25(OH)D2-S and 25(OH)D3-S). A comparative evaluation showed that the ionization efficiencies of underivatized forms in negative ion mode electrospray ionisation (ESI) are superior to those of the derivatized (using 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD)) forms in positive ion mode ESI. Separation was optimised to minimise co-elution with endogenous matrix compounds, thereby reducing ion suppression/enhancement effects. Isotopically labelled analogues of each compound were used as internal standards to correct for ion suppression/enhancement effects. The method was validated and then applied for the analysis of breastmilk and human serum. The detection limits, repeatability standard deviations, and recoveries ranged from 0.20 to 0.28fmol, 2.8 to 10.2%, and 81.1 to 102%, respectively.

While both fat- and water-soluble forms of vitamin D have been reported in diverse biological fluids, clinical and nutritional attentions have been primarily given to the fat-soluble forms [3], [4]. Therefore, clinical studies on water-soluble Vitamin D compounds are lacking. It has been suggested that the water-soluble forms (sulfate conjugates) of vitamin D have potencies similar to those of the fat-soluble compounds [5]. Although the biosynthesis of the sulfates is unclear [6], a study has shown that vitamin D is not readily sulfated in man, indicating its formation from a conjugated precursor is possible [7]. The presence of 7-dehydrocholesterol-sulfate (7-DHC-S) has been reported in human and rat skin tissue, confirming the existence of a precursor for vitamin D3-sulfate (D3-S) [6]. While controversy exists regarding the specific actions and biological roles [8], it has been reported that vitamin D-sulfate (D3-S), when orally administered in high doses, increases calcium transportation in young rats [9].

25-Hydroxyvitamin D3-sulfate (25(OH)D3-S) is a major circulating form of vitamin D and its levels in human blood may exceed those of the non-sulfated form, 25(OH)D3 [6], [7], the most commonly measured form of vitamin D to determine vitamin D status [3]. Clearly, the measurement of 25(OH)D3-S in human blood is likely to be important in the assessment of vitamin D status. 25(OH)D3-S could be considered a storage form of non-sulfated D3, as hydrolysis of the conjugate might take place in vivo [4], [6].

Vitamin D2-sulfate (D2-S) has been detected in chicken tissues and in rabbit urine [9], [10] and the biosynthesis of D2-S has been achieved in vitro [10]. It has also been demonstrated that D2-S possesses a potent antirachitic activity when administered in rats [11].

To our knowledge, the Vitamin D metabolite, 25-hydroxyvitamin D2-sulfate (25(OH)D2-S) has not yet been reported in biological fluids, and its physiological functions are not known. In fact, the vital role of D-S forms is still questioned and clear evidence for their biological function in humans has not yet been reported. A potential reason for this is the absence of a sufficiently sensitive analytical method to detect endogenous levels of these compounds.

Methods based on low sensitivity and low specificity, such as colorimetry have been reported for the analysis of D-S analogues in milk [12], [13]. Contradictory results were obtained in different studies for D-S in breastmilk [14], which may be due to the lack of specificity in analytical methods used and that saponification has been used as an extraction procedure for D-S in milk [15]. Although widely used to extract vitamin D analogues from food, saponification may be especially destructive for the water-soluble forms of this vitamin [3], [14].

Conclusions

Despite their poor sensitivities and specificities, colorimetric and LC–UV methods were previously the sole techniques available for the determination of water-soluble forms of vitamin D in milk. The currently available LC–MS/MS assay methodology is limited to the measurement of only 25(OH)D3-S in human plasma. The method presented here is a significant improvement from previous analytical methods, and one that will facilitate clinical studies of the four sulfated forms of vitamin D.

https://pubmed.ncbi.nlm.nih.gov/26708628/

2016 (8)

A Method for Simultaneous Determination of 25-Hydroxyvitamin D3 and Its 3-Sulfate in Newborn Plasma by LC/ESI-MS/MS after Derivatization with a Proton-Affinitive Cookson-Type Reagent

A method for the simultaneous determination of 25-hydroxyvitamin D3 [25(OH)D3] and its 3-sulfate [25(OH)D3S] in newborn plasma, which is expected to be helpful in the assessment of the vitamin D status, using stable isotope-dilution liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has been developed and validated. The plasma was pretreated based on the deproteinization and solid-phase extraction, then subjected to derivatization with 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD). The derivatization enabled the accurate quantification of 25(OH)D3 without interference from 3-epi-25(OH)D3 and also facilitated the simultaneous determination of the two metabolites by LC/positive ESI-MS/MS. Quantification was based on the selected reaction monitoring with the characteristic fragmentation of the DAPTAD-derivatives during MS/MS. This method was reproducible (intra- and inter-assay relative standard deviations of 7.8% or lower for both metabolites) and accurate (analytical recovery, 95.4–105.6%). The limits of quantification were 1.0 ng/mL and 2.5 ng/mL for 25(OH)D3 and 25(OH)D3S, respectively, when using a 20-μL sample. The developed method was applied to the simultaneous determination of plasma 25(OH)D3 and 25(OH)D3S in newborns; it was recognized that the plasma concentration of 25(OH)D3S is significantly higher than that of 25(OH)D3, and preterm newborns have lower plasma 25(OH)D3S concentrations than full-term newborns.

25-Hydroxyvitamin D3 3-sulfate [25(OH) D3S], the sulfated conjugate of 25(OH) D3, is another major metabolite of vitamin D3, and its circulating level was found to be much higher than that of 25(OH) D3 in infants.(5) 25(OH) D3S might be the storage form of vitamin D3; 25(OH) D3S may be utilized after deconjugation to 25(OH) D3.( 5–7) ­Therefore, it is expected that the simultaneous determination of 25(OH) D3 and 25(OH) D3S in plasma/serum is also helpful in the assessment of the vitamin D status and diagnosis for vitamin D deficiency/insufficiency of newborns/infants.

Simultaneous determination of newborn plasma 25(OH)D3 and 25(OH)D3S

The 25(OH)D3 and 25(OH)D3S concentrations in the newborn plasma were simultaneously determined based on the developed method (Fig. 4). The plasma concentration of 25(OH)D3S was 30.1±12.2 ng/mL (mean±S.D., n=59) with the range of 3.9–65.1 ng/mL and significantly higher than that of 25(OH)D3 (7.1±3.0 ng/mL, 2.7–17.0 ng/mL); these agreed with previously reported results.5) As mentioned in the introduction, 25(OH)D3S might be the storage form of vitamin D3 and utilized after deconjugation to 25(OH)D3.5–7)

Based on this concept, the simultaneous determination of the plasma 25(OH)D3 and 25(OH)D3S will be more helpful than the determination of 25(OH)D3 alone in the assessment of the vitamin D status and diagnosis for vitamin D deficiency/insufficiency of newborns. The preterm newborns have lower plasma 25(OH)D3S concentrations (Pearson’s correlation coefficient test, p<0.01), whereas the plasma 25(OH)D3 concentration was not related to the gestational age (p=0.36). The low level of 25(OH)D3S may be a possible cause of rickets that is more common in the preterm newborns. The anonymized samples were examined in this study; no subject information other than the gestational age was provided. Therefore, the gender differences in the plasma concentrations of the vitamin D3 metabolites were not examined in this study. 

Using the developed method, the concentrations of 25(OH)D3 and 25(OH)D3S in the cord plasma were also determined and compared with those in the plasma of newborns of 0 or 1 day old (n=13). For both metabolites, there were good correlations in the concentrations between the newborn plasma and cord plasma as shown in Fig. 5 (Pearson’s correlation coefficient test, p<0.01). This result indicates that the cord plasma can also be used as the specimen for the assessment of the vitamin D status for newborns.

CONCLUSION

We have developed the stable isotope-dilution LC/ESI-MS/MS method for the simultaneous determination of 25(OH)D3 and 25(OH)D3S in newborn plasma. The method employed the DAPTAD-derivatization, which enabled the accurate quantification of 25(OH)D3 without interference from 3-epi-25(OH)D3 and also facilitated the simultaneous determination of the two metabolites by positive ESI-MS/MS. The method was able to quantify 1.0–50 ng/mL of 25(OH)D3 and 2.5–50 ng/mL of 25(OH)D3S with satisfactory accuracy and reproducibility using a 20-μL plasma sample. The developed method was successfully applied to the analysis of newborn plasma; it was recognized that the plasma concentration of 25(OH)D3S is significantly higher than that of 25(OH)D3 in newborns, and preterm newborns have lower plasma 25(OH)D3S concentrations than full-term newborns. Thus, this well-characterized method will prove helpful in the assessment of the vitamin D status for newborns.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5023818/

2017 (7)

Sulfation of vitamin D3 -related compounds-identification and characterization of the responsible human cytosolic sulfotransferases

While 25-hydroxyvitamin D3 3-O-sulfate is known to be present in circulation, how it is generated in the body remains unclear. This study aimed to investigate its sulfation in major human organs and to unveil the responsible cytosolic sulfotransferases (SULTs). Of the vitamin D3 -related compounds tested, 25-hydroxyvitamin D3 and 7-dehydrocholesterol are preferentially sulfated by human organ cytosols. Among the 13 human SULTs, SULT2A1 shows sulfating activity toward all vitamin D3 -related compounds, whereas SULT1A1 and SULT2B1a/SULT2B1b show sulfating activity exclusively for, respectively, calcitriol and 7-dehydrocholesterol. These findings suggest that the metabolic pathway leading to the formation of 25-hydroxyvitamin D3 3-O-sulfate may be mediated by the sulfation of 25-hydroxyvitamin D3 or by the conversion of 7-dehydrocholesterol-3-O-sulfate in the skin.

Interestingly, 3-O-sulfated form of 25-OH-Vitamin D3 (25-OH-vitamin D3 3-O-sulfate) has been detected in human samples at similar or greater levels (ranging 10–50 nm in plasma) than unconjugated 25-OH-vitamin D3 [8-10]. Compared with that in adult plasma, a greater proportion of 25-OH-vitamin D3 3-O-sulfate (more than three times of the concentration of 25-OH-vitamin D3) has been reported to be present in infant plasma, implying the importance of sulfation in the metabolism of vitamin D3 in infants [11]. It has been proposed that, much like estrone 3-O-sulfate being a storage form of estrone [10-12], 25-OH-vitamin D3 3-O-sulfate may represent a storage form of 25-OH-vitamin D3. Moreover, 7-dehydrocholesterol 3-O-sulfate and vitamin D3 3-O-sulfate have been also detected in the biological fluids of humans and rats [10, 13]. While it is possible that sulfation may play an important role in the homeostasis of vitamin D3, the mechanism underlying the formation of sulfated vitamin D3-related compounds has remained unknown [8].

Kidney cytosol showed moderate sulfating activity toward 7-dehydrocholesterol, 25-OH-vitamin D3, and calcitriol. In contrast, skin cytosol showed only weak sulfating activity toward 7-dehydrocholesterol and calcitriol. It is noted that compared with the other three compounds, vitamin D3 was sulfated at a lower level and only by liver and small intestine cytosols. These results indicated that liver and small intestine are likely the main organs where the sulfation of 7-dehydrocholesterol, 25-OH-vitamin D3, and calcitriol may take place.

Sulfation of vitamin D3-related compounds by purified human SULTs

A systematic survey was performed in order to identify human SULTs that are capable of catalyzing the sulfation of 7-dehydrocholesterol, vitamin D3, 25-hydroxy vitamin D3, and/or calcitriol. Results indicated that SULT2A1 displayed sulfating activity toward all four vitamin D3-related compounds tested as substrates (Table 2). Notably, the vitamin D3-sulfating activity of SULT2A1 was much lower than the activities toward other substrates. In contrast, SULT1A1 showed sulfating activity toward only calcitriol, whereas SULT2B1a and SULT2B1b showed sulfating activity only with 7-dehydrocholsterol. All other SULTs showed no activity toward any of the vitamin D3-related compounds tested.

In the liver and small intestine, SULT2A1 is likely the enzyme responsible for the sulfation of 7-dehydrocholesterol due to its strong expression in these two organs [30, 32]. Whether the circulating 7-dehydrocholesterol 3-O-sulfate produced from the liver and small intestine may indeed be converted to vitamin D3 3-O-sulfate still awaits further investigation. A previous study has shown that vitamin D3 3-O-sulfate, transformed from 7-dehydrocholesterol 3-O-sulfate in the skin, may be further metabolized to form 25-OH-vitamin D3 3-O-sulfate in the liver [13]. It is noted, however, that 25-OH-vitamin D3 could also be sulfated by liver and small intestine cytosols at nearly the same level as 7-dehydrocholesterol (Table 1). In contrast, vitamin D3 was sulfated to a much lower extent by liver and small intestine cytosols. The main pathway leading to the production of 25-OH-vitamin D3 3-O-sulfate may therefore be the direct sulfation of 25-OH-vitamin D3 and/or sequential hydroxylations of 7-dehydrocholsterol 3-O-sulfate. 1α, 25-dihydroxyvitamin D3 (calcitriol), an active form of vitamin D3 produced mainly in the kidney, could also be sulfated by all five organ cytosols with liver and small intestine cytosols displaying the strongest activities. Although there is currently no report showing the presence of sulfated calcitriol in vivo, such a sulfation pathway, if confirmed, may play a role in regulating the physiological activity of calcitriol.

Results

Sulfation of vitamin D3-related compounds by human organ samples

Cytosols prepared from major human organs were tested in the enzymatic assay to examine the presence of sulfating activity toward vitamin D3-related compounds. Of the five human organ cytosols tested, liver and small intestine cytosols showed stronger sulfating activity than the other three toward all substrates tested (Table 1). Kidney cytosol showed moderate sulfating activity toward 7-dehydrocholesterol, 25-OH-vitamin D3, and calcitriol. In contrast, skin cytosol showed only weak sulfating activity toward 7-dehydrocholesterol and calcitriol. It is noted that compared with the other three compounds, vitamin D3 was sulfated at a lower level and only by liver and small intestine cytosols. These results indicated that liver and small intestine are likely the main organs where the sulfation of 7-dehydrocholesterol, 25-OH-vitamin D3, and calcitriol may take place.

Discussion

An increasing body of research findings has implicated sulfation as an important pathway involved in the metabolism of vitamin D3-related compounds [8-11]. No information, however, is currently available concerning the enzymatic mechanisms underlying the sulfation of these important biomolecules. The present study aimed to bridge this significant gap by identifying the responsible SULT enzymes and characterizing the properties of these enzymes in mediating the sulfation of vitamin D3-related compounds. Based on the results derived from this study, the sulfation pathway in the metabolism of vitamin D3-related compounds can be proposed as depicted in Fig. 3. 7-Dehydrocholesterol is a biosynthetic precursor of vitamin D3 in the skin [1, 3, 5]. A previous study has demonstrated that 7-dehydrocholesterol 3-O-sulfate can be converted to vitamin D3 3-O-sulfate in the skin [13]. While the skin cytosol tested in the present study showed significant 7-dehydrocholesterol-sulfating activity, the activity was much lower than those detected for the cytosols of liver and small intestine (Table 1). Therefore, 7-dehydrocholesterol 3-O-sulfate may be produced de novo not only in the skin but also in liver and small intestine. It is possible that the bulk of 7-dehydrocholesterol 3-O-sulfate circulating in blood may be produced in the liver and small intestine. The enzymes responsible for the sulfation of the 7-dehydrocholesterol were identified to be SULT2A1 and SULT2B1b based on the enzymatic assay (Tables 1 and 2). In the skin, SULT2B1b may play a major role in the sulfation of 7-dehydrocholesterol due to its lower Km value and the lack of SULT2A1 expression [30, 31]. In the liver and small intestine, SULT2A1 is likely the enzyme responsible for the sulfation of 7-dehydrocholesterol due to its strong expression in these two organs [30, 32]. Whether the circulating 7-dehydrocholesterol 3-O-sulfate produced from the liver and small intestine may indeed be converted to vitamin D3 3-O-sulfate still awaits further investigation. A previous study has shown that vitamin D3 3-O-sulfate, transformed from 7-dehydrocholesterol 3-O-sulfate in the skin, may be further metabolized to form 25-OH-vitamin D3 3-O-sulfate in the liver [13]. It is noted, however, that 25-OH-vitamin D3 could also be sulfated by liver and small intestine cytosols at nearly the same level as 7-dehydrocholesterol (Table 1). In contrast, vitamin D3 was sulfated to a much lower extent by liver and small intestine cytosols. The main pathway leading to the production of 25-OH-vitamin D3 3-O-sulfate may therefore be the direct sulfation of 25-OH-vitamin D3 and/or sequential hydroxylations of 7-dehydrocholsterol 3-O-sulfate. 1α, 25-dihydroxyvitamin D3 (calcitriol), an active form of vitamin D3 produced mainly in the kidney, could also be sulfated by all five organ cytosols with liver and small intestine cytosols displaying the strongest activities. Although there is currently no report showing the presence of sulfated calcitriol in vivo, such a sulfation pathway, if confirmed, may play a role in regulating the physiological activity of calcitriol.

An important issue is with regard to the functional relevance of the sulfation of the vitamin D3-related compounds. SULT-mediated sulfation is generally known to lead to the inactivation and/or the facilitated excretion of substrate compounds from the body [16-18]. Studies have shown that the sulfation of estrone forming estrone 3-O-sulfate, a main circulating form of estrone, plays an important role in the homeostasis of estrone [12, 43]. As an inactive form of estrone, estrone 3-O-sulfate serves as a precursor of estrone, and can be converted to estrone in target cells under the action of steroid sulfatase [12, 43]. It is possible that 25-OH-vitamin D3 3-O-sulfate may similarly serve as a storage form of 25-OH-vitamin D3, although the enzymatic hydrolysis of 25-OH-vitamin D3 3-O-sulfate is yet to be confirmed.

In summary, the present study demonstrates unequivocally the sulfating activities of human organ cytosols toward vitamin D3-related compounds, including 7-dehydrocholesterol, vitamin D3, 25-OH-vitamin D3, and calcitriol. The SULT enzymes responsible for the sulfation of respective vitamin D3-related compounds were identified. Of the four vitamin D3-related compounds, 7-dehydrocholesterol and 25-OH-vitamin D3 appeared to be better substrates for sulfation than the other two. Sulfation of 7-dehydrocholesterol in the skin likely occurs under the action of SULT2B1b, and sulfated 7-dehydrocholesterol may be further converted to 25-OH-vitamin D3 3-O-sulfate, a major circulating form of vitamin D3. Alternatively, 25-OH-vitamin D3 3-O-sulfate may be generated in the liver by direct sulfation of 25-OH-vitamin D3 by SULT2A1.

https://febs.onlinelibrary.wiley.com/doi/10.1002/1873-3468.12767

2017 (8)

Simultaneous quantification of 25-hydroxyvitamin D3-3-sulfate and 25-hydroxyvitamin D3-3-glucuronide in human serum and plasma using liquid chromatography-tandem mass spectrometry coupled with DAPTAD-derivatization

25-hydroxyvitamin D3-3-sulfate (25-OHD3-S) and 25-hydroxyvitamin D3-3-glucuronide (25-OHD3-G) are major conjugative metabolites of vitamin D3 found in the systemic circulation and potentially important reservoirs for 25-hydroxyvitamin D3. Simultaneous and accurate quantification of these metabolites could advance assessment of the impact of vitamin D3 on health and disease.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5585037/

2017

This thesis describes the development and validation of an LC-MS/MS method for the quantitative analysis of 12 vitamin D compounds, specifically: D2, D3, 25(OH)D2, 25(OH)D3, 24,25(OH)2D2, 24,25(OH)2D3, 1,25(OH)2D2, 1,25(OH)2D3, D3-S, D2-S, 25(OH)D3-S and 25(OH)D2-S in biological fluids.

The biological roles of vitamin D-sulfate compounds (D3-S, D2-S, 25(OH)D3-S and 25(OH)D2-S) are unclear to date, probably due to the lack of sufficiently sensitive assay methods to study them. It has been suggested that sulfated compounds are the storage forms of the non-conjugated metabolites, and may have similar potencies.

1.3.5 25(OH)D3-Sulfate, 25(OH)D2-Sulfate, D3-Sulfate and D2-Sulfate

In 1985, one study first identified the existence of 25-hydroxyvitamin D3-sulfate [25(OH)D3S] in human plasma (77). Although the biological role of 25(OH)D3-S is still not fully understood, there is an assumption that it might be a storage form of vitamin D3(78, 79). The circulating levels of this metabolite were found to be similar to or exceeding that of the major circulating form (25(OH)D3) in adults and infants, respectively (77, 80). Therefore, it has been suggested that conjugation of vitamin D with sulphate plays an important role in vitamin D metabolism. It is also considered that the quantification of 25(OH)D3-S in plasma/serum was viewed as being important in researching its significance in pregnant women and newborns as well as for its assessment of vitamin D status, especially in infants (78). Sasashi et al. suggested that vitamin D2-S has approximately the same antirachitic potency as vitamin D2(81). Miravet et al. further reported potent biological activity of the vitamin D3- Sand D3 (82).However, the results from a study by Cancela et al. shows that vitamin D3-S is clearly less active than the same dose of free vitamin D3 in promoting normal mineral homeostasis and bone mineralisation in rats during the lactation period(83). In 2015, a method using LC with MS detection has been reported for the quantification of four sulfated forms of vitamin D: vitamins D2 and D3-sulfate (D2-S and D3-S) and 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3-sulfate (25(OH)D2-S and 25(OH)D3-S) in human serum and breastmilk (84). Figure 1.5 shows the chemical structures for vitamin D-sulfate.

Of the four known sulfated conjugates, vitamin D-sulfate (D3-S and D2-S) and 25-hydroxyvitamin D-sulfate (25(OH)D3-S and 25(OH)D2-S), 25(OH)D3-S has been the only form found in human and animal blood to date (77). The circulating levels of this compound were found to be similar to or higher than those of 25(OH)D3 in adults and infants, respectively (19, 77, 80). Since the level of 25(OH)D3S is similar to or exceeds that of the major circulating form of vitamin D in adults, it was viewed as being important in researching the significance of the sulfate metabolite in pregnant women and newborns (80). Despite being a major circulating form of vitamin D in man the biological role of 25(OH)D3- S is not yet known, and has been therefore, considered to be a “storage” form of the major circulating form 25(OH)D3 (77). The lack of research into the biological function of 25(OH)D3-S may be due to the scarcity of the methods available for its accurate quantification, until recently, for its accurate quantification (78). In studies where up to six analogues and two epimers of vitamin D were determined simultaneously, sulfate metabolites were not included (204)

While controversy exists regarding their biological roles, it has been reported that 25-hydroxy D3-sulfate [25(OH)D3-S] may be a storage form of vitamin D3 and knowledge of its concentration may be expected to be helpful in the assessment of the vitamin D status, especially that of infants (78).

Vitamin D-sulfates are acidic in nature due to the presence of the readily ionisable sulfate moiety, and are capable, by deprotonation, of producing negatively charged ions.

This study is the first to report the development and validation of an analytical method for the simultaneous detection and quantification of 12 vitamin D compounds (both lipophilic and hydrophilic) in human serum. The high accuracy, selectivity and sensitivity obtained demonstrate the suitability of the proposed method for the determination of vitamin D compounds and their metabolites in man, that are potentially useful in disease diagnosis.

To our knowledge, this study was the first to attempt to detect and quantitate vitamin D sulphate metabolites in mouse brain; we observed, however, that sulphate forms were not present in any of the samples analysed. We consider that the absence of D2-S, D3-S, 25(OH)2D3-S and 25(OH)2D2-Sis due to the potential impact of the blood-brain barrier (BBB) on the transportation of sulfate metabolites into the cerebrospinal fluid. The BBB is a physical and biochemical barrier between the blood and the brain, which prevents entry into the brain of most drugs and endogenous compounds from the blood. Only small lipophilic compounds can diffuse passively through the BBB, while other compounds  are usually able to cross the BBB only with the help of carrier proteins (275). Our results showed that membrane vitamin D3 receptors have also been identified in the brain and the concentrations of 1,25(OH)2D3 in the mouse brain tissue samples is markedly higher than that of other metabolites. This metabolite is able to cross the blood-brain barrier and bind to nuclear vitamin D3 receptors in the brain (276, 277). In comparison to human serum concentrations, 1,25(OH)2D3levels in mouse brain were observed to be greater. It has been shown that a physiological concentration of the concentration in brain tissue of 25(OH)D3 was five percent or less than that of 1,25(OH)2D3 due to their polarity differences (278). Owing to the added sulfate group, the polarities of D2-S, D3-S, 25(OH)D3-S and 25(OH)D2- S are likely greater than the polarity of lipophilic compounds – this is seen with other sulphate metabolites (279). Thus, it might be expected that the concentrations of vitamin D-sulfate compounds would be much reduced or indeed undetectable. This is possibly one of the main reasons that vitamin D-sulfate levels in brain tissue are largely unexplored and almost never measured in clinical practice.

https://espace.library.uq.edu.au/data/UQ_728352/s43715150_final_thesis.pdf

2018 (4)

Polymorphic Human Sulfotransferase 2A1 Mediates the Formation of 25-Hydroxyvitamin D3-3-O-Sulfate, a Major Circulating Vitamin D Metabolite in Humans

Metabolism of 25-hydroxyvitamin D3 (25OHD3) plays a central role in regulating the biologic effects of vitamin D in the body. Although cytochrome P450–dependent hydroxylation of 25OHD3 has been extensively investigated, limited information is available on the conjugation of 25OHD3. In this study, we report that 25OHD3 is selectively conjugated to 25OHD3-3-O-sulfate by human sulfotransferase 2A1 (SULT2A1) and that the liver is a primary site of metabolite formation. At a low (50 nM) concentration of 25OHD3, 25OHD3-3-O-sulfate was the most abundant metabolite, with an intrinsic clearance approximately 8-fold higher than the next most efficient metabolic route. In addition, 25OHD3 sulfonation was not inducible by the potent human pregnane X receptor agonist, rifampicin. The 25OHD3 sulfonation rates in a bank of 258 different human liver cytosols were highly variable but correlated with the rates of dehydroepiandrosterone sulfonation. Further analysis revealed a significant association between a common single nucleotide variant within intron 1 of SULT2A1 (rs296361; minor allele frequency = 15% in whites) and liver cytosolic SULT2A1 content as well as 25OHD3-3-O-sulfate formation rate, suggesting that variation in the SULT2A1 gene contributes importantly to interindividual differences in vitamin D homeostasis. Finally, 25OHD3-3-O-sulfate exhibited high affinity for the vitamin D binding protein and was detectable in human plasma and bile but not in urine samples. Thus, circulating concentrations of 25OHD3-3-O-sulfate appear to be protected from rapid renal elimination, raising the possibility that the sulfate metabolite may serve as a reservoir of 25OHD3 in vivo, and contribute indirectly to the biologic effects of vitamin D.

25OHD3-3-O-sulfate is reported to be a major circulating metabolite of 25OHD3 in humans, with an average circulating concentration comparable to that of 25OHD3 (Axelson, 1985; Shimada et al., 1995; Higashi et al., 2014), and thus, the sulfonation metabolic pathway might contribute importantly to vitamin D homeostasis. Surprisingly little is known about how and where 25OHD3-3-O-sulfate is formed. This information is crucial if the contribution from the sulfonation pathway to vitamin D homeostasis is to be fully evaluated. In this study, we examined the sulfonation of 25OHD3 in humans using recombinant sulfotransferase (SULT) enzymes, liver cytosols, primary hepatocytes, renal tubular epithelial cells, and immortalized intestinal epithelial cells to identify which of the human SULTs is responsible for the sulfonation reaction and in what tissues the reaction occurs, as well as potential genetic and environmental sources of interindividual variability in hepatic sulfonation activity.

The formation of 25OHD3-3-O-sulfate from 25OHD3 could be considered a catabolic process, but some investigators have hypothesized that it also represents an alternative 25OHD3 storage form in the body (Higashi et al., 2010). This is quite plausible because other endogenous steroid sulfate conjugates, such as estradiol-sulfate and dehydroepiandrosterone (DHEA)-sulfate, circulate at relatively high levels, are deconjugated in target tissues, and contribute to certain physiologic functions (Axelson, 1987; Banerjee et al., 2013; Sánchez-Guijo et al., 2015). Similarly, 25OHD3-3-O-sulfate might be retained in the circulation and distributed to different tissues of the body where it could be hydrolyzed to 25OHD3, replenishing the 25OHD3 pool, as needed. With this in mind, we also tested the binding affinity of 25OHD3-3-O-sulfate for DBP and its presence in human urine and bile.

These results suggest that SULT2A1 is the predominant source of sulfonation activity in the human liver and could represent an important metabolic route for 25OHD3 clearance.

25OHD3-3-O-Sulfate Is the Major Metabolite Formed from 25OHD3 by Human Hepatocytes

Previous studies have demonstrated that at nonphysiologic concentrations (1–5 µM), 25OHD3 is metabolized to 1α,25(OH)2D3, 24R,25(OH)2D3, 4α,25(OH)2D3, 4β,25(OH)2D3, 25OHD3-3-O-glucuronide, 25OHD3-25-O-glucuronide, and putative 5,6-trans-25OHD3-25-O-glucuronide when incubated with human hepatocytes (Wang et al., 2014). However, formation of 25OHD3-3-O-sulfate in human hepatocytes was not determined. For this investigation, a more physiologic concentration of 25OHD3 (50 nM) was applied to cultured human hepatocytes to generate a metabolite profile. As shown in Fig. 3A, all major metabolites of 25OHD3, except 1α,25(OH)2D3, were detected in the incubations. 25OHD3-3-O-sulfate was the most abundant product observed, followed by 24R,25(OH)2D3, 4,25(OH)2D3 [4α,25(OH)2D3 and 4β,25(OH)2D3], and 25OHD3-glucuronides. Formation of these 25OHD3 metabolites occurred in a linear, time-dependent manner, except for 4β,25(OH)2D3 and 4α,25(OH)2D3 formation, which underwent extensive sequential glucuronidation, as previously reported (Wang et al., 2013a).

By comparison, renal tubule epithelial cells and LS180 intestinal epithelial cells showed no detectable formation of 25OHD3-3-O-sulfate during a 24-hour incubation with 50 nM 25OHD3 (data not shown). These cells have previously been shown to catalyze the 24-hydroxylation of 25OHD3 under similar culture conditions (Zheng et al., 2012; Weber et al., 2016). Based on the assay limit of detection, culture conditions, and the approximate number of cells per well, it was estimated that 25OHD3 sulfonation activity per renal tubule epithelial cell or LS180 cell was <10% that of cryopreserved human hepatocytes.

VDBP

Binding of 25OHD3-3-O-Sulfate to Rat Plasma DBP

The presence of 25OHD3-3-O-sulfate in plasma, but not urine, suggested the possibility that it might have significant binding affinity for the DBP, restricting its excretion by the kidney, as occurs for 25OHD3. Previous studies have shown that 25OHD3-3-O-glucuronide, but not 25OHD3-25-O-glucuronide, binds tightly to DBP (Wang et al., 2014). Due to the structure similarity, we surmised that 25OHD3-3-O-sulfate could also bind to DBP. To test this hypothesis, the concentration-dependent binding of 25OHD3-3-O-sulfate, 25OHD3-3-O-glucuronide, and 25OHD3 to rat plasma DBP was measured by radioligand binding assay (Horst et al., 1981). As seen in Fig 7. the binding affinity of 25OHD3-3-O-sulfate for DBP was essentially identical to that of 25OHD3 and 25OHD3-3-O-glucuronide. The mean EC50 values for 25OHD3, 25OHD3-3-O-glucruonide, and 25OHD3-3-O-sulfate binding to DBP under current incubation conditions were 0.82, 0.74, and 0.72 pmol, respectively.

Binding of 25OHD3-3-O-Sulfate and 25OHD3 to the DBP

The binding assays were performed as described previously (Horst et al., 1981). Briefly, rat plasma was diluted 1:5000 (v/v) in 0.05 M phosphate buffer (pH 7.5) containing 0.01% gelatin and 0.01% merthiolate (PBG buffer). Each assay mixture was placed in a borosilicate glass tube and consisted of the following: 1) 0.5 ml 1:5000 dilution of vitamin D–deficient rat plasma in PBG buffer, 2) 6000–8000 cpm [23,24-3H]-25OHD3 in 20 µl 100% ethanol, and 3) vitamin D metabolites in 25 µl ethanol. After 1 to 2 hours of incubation at 4°C, the bound vitamin D metabolites were separated from free vitamin D metabolites by adding 0.2 ml of a mixture of cold 1.0% Norit A Charcoal and 0.1% Dextran T-70 (Sigma-Aldrich, St Louis, MO) in PBG buffer to each tube. After 30 minutes at 4°C, the tubes were spun at 1000g for 10 minutes in a refrigerated centrifuge. A portion (0.5 ml) of the supernatant was removed for quantitation of the bound [3H]-25OHD3. The binding experiment was conducted with rat plasma so that we could directly compare new results to historical data generated by our laboratory using rat plasma (Wang et al., 2014). Previously, Bouillon et al. (1980) concluded that the heterogeneity between the genes encoding for DBP in rats and humans, and the subsequent structural difference between the two proteins, has no effect on their affinity or capacity to bind vitamin D metabolites.

Herein, we confirm that 25OHD3-3-O-sulfate is also a quantitatively important circulating product of 25OHD3 (Axelson, 1985; Shimada et al., 1995) and we report for the first time that it is generated primarily in the liver by the enzyme SULT2A1. The sulfate conjugate was the dominant metabolic product of 25OHD3 produced by primary human hepatocytes when incubated with a physiologically relevant 50 nM concentration of 25OHD3. Thus, interindividual differences in sulfonation activity may be a major source of variation in circulating blood 25OHD3 concentrations (Fig 8)., assuming that hepatic clearance of 25OHD3 is an important determinant of 25OHD3 accumulation in the body.

Results from our kinetic experiments, revealing comparable Km and Vmax values when normalized for SULT2A1 content, suggest that this isozyme is the predominant catalyst of the 25OHD3 sulfonation reaction in the human liver. This conclusion is supported in part by the positive regression of liver cytosolic sulfonation activity against SULT2A1 protein content and the strong association between SULT2A1 genetic variation and liver cytosolic sulfonation activity. However, we note that approximately one-half of the variance in liver cytosolic 25OHD3 sulfonation activity remained unexplained and that variation in cytosolic SULT2A1 protein content explained only 21% of the unadjusted metabolic activity toward this substrate, in contrast to a much stronger correlation between cytosolic DHEA sulfonation activity and SULT2A1 protein content. Nonspecific factors associated with the liver procurement site explained a substantial fraction of the observed variance in liver cytosolic 25OHD3 sulfonation activity and variable storage time and possible loss of activity but not measured protein was also contributory (Table 2). We speculate that these and possibly other factors (e.g., uncontrolled substrate-selective protein-SULT2A1 interactions) masked what would otherwise have been a stronger association between SULT2A1 protein level and sulfonation activity.

SULT2A1 is known to metabolize hydroxysteroids, such as estradiol, DHEA and bile acids (Chatterjee et al., 2005). That it also catalyzes 25OHD3 sulfonation is not surprising, considering structural similarities. The regiospecificity of SULT2A1 toward 25OHD3 sulfonation, almost exclusively at the 3-position, is similar to that observed for hydroxysteroids and oxysterols. Interestingly, the related SULT isoform, SULT2B1, showed little activity toward 25OHD3, which is in contrast to its ability to generate sulfate metabolites of hydroxysteroids and oxysterols that are substrates for SULT2A1 (Falany and Rohn-Glowacki, 2013). SULT2B1 is expressed primarily in extrahepatic tissues (Falany and Rohn-Glowacki, 2013), whereas SULT2A1 is highly expressed in the liver and adrenal cortex and less so in the gastrointestinal tract (Chatterjee et al., 2005). This distribution pattern suggests that there will be limited 25OHD3 sulfonation activity outside of the liver. Consistent with these findings, no detectable 25OHD3 sulfonation was observed in either human intestinal LS180 cells or renal epithelial cells incubated with a physiologically relevant concentration of 25OHD3.

It is noteworthy that SULT2A1 is thought to be regulated by 1α,25(OH)2D3 (Echchgadda et al., 2004), suggesting an autofeedback mechanism of vitamin D regulation, as described for CYP24 (Haussler et al., 2013) and proposed for CYP3A4 (Xu et al., 2006; Wang et al., 2013a).

25OHD3-3-O-sulfate was found to have a high binding affinity for DBP, which explains its relatively high abundance in plasma and absence from urine. The crystal structure of DBP indicates that the vitamin D binding site is a cleft, which can easily accommodate large substituents at the C-3 position of 25OHD3 (e.g., conjugated moieties) (Verboven et al., 2002). High binding affinity to DBP would reduce the renal excretion of 25OHD3-3-O-sulfate into urine. We speculate that the complex of 25OHD3-3-O-sulfate and DBP is filtered and then reabsorbed in renal proximal tubules by megalin/cubilin-mediated endocytosis, as shown for the 25OHD3-DBP complex (Rowling et al., 2006).

Although 25OHD3-3-O-sulfate is a major circulating form of vitamin D3, whether it possesses biologic activity directly or indirectly is unclear. A number of studies have been conducted to understand the biologic activities of vitamin D3-sulfate, a conjugated metabolite of vitamin D3 (Higaki et al., 1965; Sahashi et al., 1967a,b, 1969). Vitamin D3-sulfate was synthesized previously (Reeve et al., 1981) and its biologic activity was determined in a vitamin D–deficient rat model. Activity was observed, but only at doses higher than what can be elicited by vitamin D3 (Nagubandi et al., 1981). Later studies also showed less biologic activity of vitamin D3-sulfate than free vitamin D3 in vivo (Cancela et al., 1985). However, in each of these studies, vitamin D3-sulfate was administered, rather than having the metabolite generated in situ, with the uncertainties of bioavailability and access to cellular sites that complicate quantitative comparisons. Thus, it is possible that 25OHD3-3-O-sulfate might undergo hydrolysis, catalyzed by ubiquitous sulfatases and regenerate 25OHD3. This type of hormone conjugate cycling is observed for estrogen and DHEA (Mueller et al., 2015), where the sulfo-conjugates are the dominant form in blood circulation and are distributed to peripheral tissues where desulfonation can occur. In the case of DHEA-3-O-sulfate, conversion to DHEA is followed by metabolism to androstenedione and downstream androgens and estrogens (Strott, 2002).

Finally, given the detection of 25OHD3-3-O-sulfate in bile, we are intrigued by the possibility that preferential delivery of the hormone conjugate to the duodenum and upper small intestine might explain the preferential expression of vitamin D receptor target genes, such as CYP3A4, transient receptor potential cation channel subfamily V member 6 (TRPV6), and calbindin D9K, in the upper small intestine (Wang et al., 2013b). Results from unpublished studies indicate that 25OHD3-3-O-sulfate is a substrate for the cell uptake transporter, organic anion transporting polypeptide 2B1 (OATP2B1), which is expressed in the intestinal epithelia (Drozdzik et al., 2014). Once absorbed into mucosal epithelial cells, 25OHD3-3-O-sulfate could be hydrolyzed to 25OHD3 and then undergo 1α-hydroxylation to the active hormone and contribute to the regulation of TPRV6, calbindin D9K, and CYP3A4 (Wang et al., 2013b). With regard to the kidney, 25OHD3-3-O-sulfate bound to the DBP in blood could be filtered in the glomerulus and then reabsorbed in the proximal tubular epithelium through the action of megalin/cubilin, similar to what occurs for the 25OHD3-DBP complex (Negri, 2006). Again, intracellular hydrolysis of the conjugate and bioactivation to 1α,25(OH)2D3 could contribute to the known biologic effects of vitamin D in this tissue. Further work is needed to explore these mechanistic hypotheses.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5829543/

2018 (8)

SULFATION PATHWAYS: Insights into steroid sulfation and desulfation pathways

Selected steroid species in sulfo-focus

Several steroid conjugates have been known for decades, but only recently have these forms been thought to be biologically meaningful and worth studying. Here, we briefly review knowledge about vitamin D sulfates, steroid disulfates and 11-oxo-androgens.

Vitamin D

25-Hydroxy-vitamin D3-3-sulfate (25-OH-D3-S) is a major metabolite of vitamin D3 found in the systemic circulation (Axelson 1985). As circulating concentrations of 25OH-D3-3-O-sulfate seem not to be rapidly secreted by the kidney, there is the possibility that this sulfate metabolite may serve as a reservoir of 25OH-D3 in vivo, contributing indirectly to the biological effects of vitamin D (Wong et al. 2018). Sulfotransferase SULT2A1 was identified as the major vitamin D3-sulfating enzyme (Kurogi et al. 2017, Wong et al. 2018). SULT2A1 showed activity toward several vitamin D3-related compounds, whereas SULT1A1 and SULT2B1a/SULT2B1b only showed sulfating activity for, respectively, calcitriol and 7-dehydrocholesterol (Kurogi et al. 2017).

The relationship between vitamin D and sulfation pathways is reciprocal. The vitamin D receptor also induces transcription of the steroid sulfotransferases SULT2A1 (Echchgadda et al. 2004) and SULT2B1b (Seo et al. 2013) as well as the phase I monooxygenase CYP3A4 (Ahn et al. 2016), among other genes. Interestingly, the induction of STS by vitamin D3 and retinoids was reported in HL60 promyeloid cells (Hughes et al. 2001). As net effect, vitamin D transcriptional regulation results in androgen inactivation (Ahn et al. 2016) and elevated sulfation activity that might increase the levels of vitamin D sulfate metabolites.

Several analytical methods have been reported to detect and quantify vitamin D3 sulfoconjugates (Higashi et al. 2014, Gao et al. 2017, Abu Kassim et al. 2018). Axelson reported values of 35 ± 14 nM for 25-hydroxy-D3-3-sulfate in plasma from 60 patients (Axelson 1985), Gao measured 56 ± 24 nM for 25-OH-D3-3-sulfate in serum from six healthy volunteers (Gao et al. 2017) and Abu Kassim found a range of 9.52–43.8 nM for 25-OH-D3-3-sulfate in serum of ten volunteers (Abu Kassim et al. 2018). Concentrations of this vitamin D3 sulfoconjugate were consistently higher than its glucuronidated counterparts. More importantly, the reported circulating concentrations for vitamin D3-3-sulfate reach up to what is regarded as the normal level of circulating 25-OH-vitamin D3, 80–250 nM (Hollis 2010). Early studies described vitamin D3-3-sulfate as less biologically active than free vitamin D3 in rodents (Nagubandi et al. 1981, Cancela et al. 1987). Considering the high circulating concentrations of 25-OH-D3-3-sulfate in the human circulation, it should be taken into account when determining a person’s vitamin D status – it could be a reservoir for local generation of 25-OH-D3 and the active 1,25-di-OH-D3.

Most researchers in this area focus on sulfated estrogens and androgen precursors (e.g. DHEAS); however, we have little grasp of whether other sulfated steroids, such as vitamin D, represent biologically relevant reservoirs for local desulfation and subsequent action.

https://jme.bioscientifica.com/view/journals/jme/61/2/JME-18-0086.xml

2018 (9)

The serum vitamin D metabolome: What we know and what is still to discover.

2.2.5. Conjugated forms of vitamin D 25-Hydroxyvitamin D3–3-sulfate (25(OH)D3–3-sulfate) has been identified as a major form of vitamin D3 in human serum with the first quantitative measurement on 10 healthy individuals giving a mean serum concentration of 46 nM [165]. The 25(OH)D3–3-sulfate concentration is higher than that of 25(OH)D3 in the fetal circulation, including at birth, where the concentration is approximately 73 nM [166, 167]. A quantitative LC/MS/MS assay for both 25(OH)D3–3-sulfate and 25(OH)D3 in newborn plasma (20 μL) has been reported using DAPTAD derivatization without interference from 3-epi25(OH)D3 [167]. A quantitative assay of four sulfated forms of vitamin D: vitamin D3–3-sulfate, vitamin D2–3-sulfate, 25(OH)D3–3-sulfate and 25(OH)D2–3-sulfate has been recently reported using LC/MS/MS with ESI in the negative ion mode, without sample derivatization [168]. All four sulfated forms were quantitated in human serum, with mean concentrations of vitamin D2–3-sulfate and vitamin D3–3-sulfate being 0.50 and 0.70 nM, respectively, and 25(OH)D2–3-sulfate and 25(OH)D3–3sulfate being 1.5 and 10.4 nM, respectively.

The cytosol from several human tissues including liver and small intestine can catalyze the 3sulfation of 25(OH)D3, with SULT2A1 identified as the major sulfotransferase involved [169]. Other sulfotransferases including SULT2B1a and SULT2B1b can 3-sulfate 7DHC, providing another possible synthetic pathway leading to vitamin D3–3-sulfate and 25(OH)D3–3-sulfate [169]. It has been proposed that 25(OH)D3–3-sulfate is a storage form of 25(OH)D3 [165] but to our knowledge measurements of its half-life in serum are yet to be made. It is likely that its hydrolysis and activation to 1α,25(OH)2D3 are necessary for any biological activity, although no direct measurement of the binding of 25(OH)D3–3-sulfate to the VDR have been made. Vitamin D3–3-sulfate was much less active on bone calcium metabolism when fed to rats compared to vitamin D3 [170]. Since SULT2A1 can act on 1α,25(OH)2D3, the resulting sulfate ester may also be present in the serum, especially in the fetus.

Recently, 25(OH)D3–3-glucuronide was measured in the serum of six healthy volunteers along with 25(OH)D3–3-sulfate, using DAPTAD sample derivatization. The reported mean concentration of 25(OH)D3–3-glucuronide was 3.4 nM with 25(OH)D3–3-sulfate being 55.6 nM [171]. Vitamin D metabolites are primarily excreted in bile as their glucuronides [155]. The 3-monoglucuronide of vitamin D3 itself has been reported to be present in bile [172] and glucuronides of 23S,25(OH)2D3, 24R,25(OH)2D3 and 24-oxo,23S,25(OH)2D3 have been detected in human urine [155], so these conjugates may also be present at low concentrations in human serum.

The relatively high concentrations of 25(OH)D3–3-sulfate in serum, particularly in infants (section 2.2.5) suggests that this metabolite should be measured to fully assess vitamin D status. Its concentrations in adults are comparable to those of 3-epi-25(OH)D3, but unlike 3-epi-25(OH)D3 it may be physiologically active in terms of being able to be converted to 1α,25(OH)2D3. However, how rapidly it can be converted to 25(OH)D3 to “buffer” 25(OH)D3 concentrations remains to be established.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6342654/

2020

Altered Vitamin D Metabolism in Health and Disease

Patients with CF had 15%, 35%, and 40% lower circulating concentrations of 1α,25(OH)2D3, 4β,25(OH)2D3, and 25(OH)D3-S, respectively.

To assess changes in 25(OH)D3 metabolism during pregnancy, we measured 25(OH)D3 and selected metabolites in a longitudinal study of healthy women (n = 15) before, during, and after pregnancy (Chapter 4). The change in the concentration of 25(OH)D3 and its metabolites were evaluated using linear mixed-effects, modeling. The model predicted increase in the concentration of 25(OH)D3 was 45% during pregnancy. Serum concentrations of 1α,25(OH)2D3, 4β,25(OH)2D3, and 25(OH)D3-G increased by 200%, 14%, and 38%, respectively, as estimated by the linear mixed-effects model from pre-pregnancy to 36 weeks of gestation (p < 0.001). Additionally, 25(OH)D3-S concentrations decreased 25% from pre-pregnancy to 36 weeks of gestation (p < 0.001 for all). Protein binding was altered during pregnancy; vitamin D binding protein (VDBP) serum concentrations increased by 190% and albumin concentrations decreased 26% from prepregnancy to 36 weeks of gestation, resulting in a 20% decrease in the percent unbound of 25(OH)D3.

1.7.5. Formation of 25-hydroxyvitamin D3-3-O-Sulfate A major metabolite of 25(OH)D3 is 25-hydroxyvitamin D3-O-sulfate (25(OH)D3-S), which circulates at plasma concentrations similar to 25(OH)D3 (10 to 40 ng/mL) (75-80). Using in vitro incubations with human liver cytosol, recombinant sulfotransferase (SULT) enzymes and human hepatocytes, investigators identified SULT2A1 as the liver enzyme responsible for the formation of 25(OH)D3-S from 25(OH)D3 (75, 81). 25(OH)D3-S was detected in human bile at a concentration of approximately 1.3 ng/mL, which supports the hypothesis that 25(OH)D3-S undergoes enterohepatic recirculation and may serve as an additional source of 25(OH)D3 once deconjugated (75). Additionally, 25(OH)D3-S circulates tightly bound to VDBP and is not excreted into the urine (75).

https://www.proquest.com/openview/f7c0958c4ceed883658068bce9927220/1

2020 (10)

3-Epi-25-hydroxyvitamin D3 is a poor substrate for SULT2A1: Analysis of its 3-sulfate in cord plasma and recombinant human SULT2A1 incubate

25(OH)D3 3-sulfate [25(OH)D3-3S, Fig. 1a] is another major circulating metabolite of vitamin D3, and its plasma level (mean, ca. 60 nM) was found to be much higher than that of 25(OH)D3 (mean, ca 20 nM) in newborns [10,11]. 25(OH)D3-3S has a high binding affinity for the vitamin D binding protein [12], which would be the main cause for its relatively high abundance in the circulation. Recently, human sulfotransferase (SULT) 2A1 in the liver was identified as the major enzyme responsible for the 3-O-sulfation of 25(OH)D3 [12,13]. 25(OH)D3-3S might be the storage form of vitamin D3 because 25(OH)D3 can be regenerated from this sulfate by deconjugation.

As just described, 25(OH)D3-3S is the most abundant circulating metabolite of vitamin D3 in newborns [10,11]. The plasma/ serum concentration ratio of Epi-25(OH)D3 to 25(OH)D3 is higher in newborns/infants than in adults [7,8]. Taken these two findings together, the question about the presence or absence of Epi-25(OH)D3 3- sulfate [Epi-25(OH)D3-3S] in the plasma/serum of newborns/infants arose. Clarifying this question could improve our understanding of the metabolism of vitamin D3.

To determine what caused this result, we next performed an in vitro experiment of the 3-O-sulfation for 25(OH)D3 and Epi-25(OH)D3 using the recombinant human sulfotransferase (SULT) 2A1. This in vitro experiment revealed that Epi-25(OH)D3 is a poor substrate for the 3-O-sulfation catalyzed by SULT2A1 as compared to 25(OH)D3. This substrate specificity of SULT2A1 would be the main cause for the result obtained from the analysis of the cord plasma samples.

Thus, a variety of metabolites derived from 25(OH)D3 is found in human plasma/serum, and it is thought that the metabolism of 25(OH)D3 plays a primary role in regulating the biological activities of vitamin D3 in humans. As just described, 25(OH)D3-3S is the most abundant circulating metabolite of vitamin D3 in newborns [10], [11]. The plasma/serum concentration ratio of Epi-25(OH)D3 to 25(OH)D3 is higher in newborns/infants than in adults [7], [8]. Taken these two findings together, the question about the presence or absence of Epi-25(OH)D3 3-sulfate [Epi-25(OH)D3-3S] in the plasma/serum of newborns/infants arose. Clarifying this question could improve our understanding of the metabolism of vitamin D3.

https://www.sciencedirect.com/science/article/abs/pii/S0039128X20301215

2021 (5)

Development of a LC-MS/MS method to measure serum 3-sulfate and 3-glucuronide 25-hydroxyvitamin D3 metabolites; comparisons to unconjugated 25OHD in pregnancy and polycystic ovary syndrome

Abstract

Vitamin D status is routinely assessed by measuring circulating concentrations of 25-hydroxyvitamin D (25OHD2 or 25OHD3). However as deconjugation is not routinely incorporated into sample treatment prior to analysis, conjugated forms of 25OHD (particularly the more abundant 25OHD3) are often not considered in determining serum concentrations of total 25OHD. Two major circulating conjugated forms of 25OHD3 are 25-hydroxyvitamin D3-3-sulfate (25OHD3-S) and 25-hydroxyvitamin D3-3-glucuronide (25OHD3-G). Incorporating these two conjugated metabolites into the measurement of vitamin D status could improve our understanding of vitamin D status in health, particularly if there are changes in sulfation and glucuronidation activities. The aim of this study was to develop a liquid chromatography tandem-mass spectrometry (LC-MS/MS) targeted method for measurement of 25OHD3-S and 25OHD3-G in serum to enable comparisons with circulating levels of the free 25OHD3 form. We developed and validated a new LC-MS/MS method that measured both 25OHD3-S and 25OHD3-G following a solid phase extraction sample preparation method. Partial separation of analytes by LC, and the separation of analytes by the optimized multiple reaction monitoring transitions enabled the quantitation of both 25OHD3-S and 25OHD3-G in the single method. Serum concentrations of 25OHD3-S (24.7 ± 11.8 ng/mL) and 25OHD3-G (2.4 ± 1.2 ng/mL) were shown to be a significant proportion of circulating vitamin D metabolites in healthy donor serums. These levels of 25OHD3-S and 25OHD3-G closely associated with 25OHD3 concentrations, r = 0.728, p = 0.001 and r = 0.632, p = 0.006 respectively. However in serum from pregnant women and non-pregnant women with polycystic ovary syndrome (PCOS) significant differences in the ratios between conjugated and free 25OHD3 were observed between pregnancy groups (25OHD3/25OHD3-S and 25OHD3/25OHD3-G p < 0.001), and between healthy and PCOS subjects (25OHD3/25OHD3-G p < 0.050). Development of this novel high-throughput LC-MS/MS method indicates that 25OHD3-S and 25OHD3-G are substantial components of circulating vitamin D metabolites. The concentrations of these metabolites relative to conventional 25OHD3 may vary in different physiological and pathophysiological settings, and may therefore play an unrecognized but important role in the actions of vitamin D.

The major circulating sulfated 25OHD3 metabolite is 25OHD3-3-sulfate (25OHD3-S) which has previously been measured in circulation at levels similar to that of 25OHD3 [12], [14], [15], [16]. Sulfotransferase SULT2A1 has been identified as the major sulfating enzyme of 25OHD3, as well as other vitamin D metabolites [17], [18], [19]. The SULT1A1 or SULT2B1a/b enzymes do not have sulfation activity towards 25OHD3, however these SULT enzymes have shown activity for sulfation of calcitriol and 7-dehydrocholesterol respectively [17], [19]. This has led to the hypothesis that the vitamin D3-S could be hydroxylated to form 25OHD3-S [19]. The sulfation rate of 25OHD3 by SULT2A1 varies between individuals based on a single nucleotide variant of SULT2A1 [18].

The biological roles of 25OHD3-G and 25OHD3-S and their contribution to 25OHD status are still not fully understood, despite 25OHD3-S being identified in circulation as early as 1985 [20]. One hypothesis suggests that reduced renal elimination enables higher concentrations of the conjugated 25OHD3 metabolites in circulation. In this way, 25OHD3-G and 25OHD3-S could serve as a reservoir for 25OHD3 through deconjugation to 25OHD3, particularly at target tissue sites [3], [12], [16], [18]. Analysis of conjugated 25OHD3 metabolites, along with free 25OHD3 measures could therefore be important in providing a more comprehensive assessment of vitamin D status, particularly in different disease states in which conjugation and hydrolysis activity may be altered.

Previous studies have reported methods using liquid chromatography (LC) coupled to ultraviolet detection (UV) or mass spectrometry (MS) for measuring 25OHD3-S [14], [15], [21], [22] or 25OHD3-G [9]. A recent method has described a simultaneous method to measure both 25OHD3-S and 25OHD3-G in circulation. However, this method required derivatization for reliable detection of the 25OHD3-G analyte [12]. There is also limited knowledge on the clinical role of vitamin D conjugation, as measurements and the ratios between conjugated 25OHD3 and the free form have not previously been reported for different health and disease groups.

https://www.sciencedirect.com/science/article/abs/pii/S0039128X21000246

2021 (5)

Vitamin D: Current Challenges between the Laboratory and Clinical Practice

“Conjugation is a mechanism that changes the solubility of compounds, which alters their biological activity and the probability of their elimination from the organism.  Sulfation is performed by the enzyme sulfotransferase (SULT), and this process was thoroughly described, inter alia, for steroidal dehydroepiandrosterone and its sulfate [35]. Vitamin D and its metabolites are typically converted by the subtype SULT2A1, and the rate of sulfation is associated with the gene variant encoding the enzyme [36]. 25(OH)D3-3-sulfate, with a mean concentration of 16.7 ng/mL, was identified as the most abundant sulfated form of vitamin D in serum, with levels often exceeding those of unconjugated 25(OH)D3 [37]. Other vitamin D sulfates (25(OH)D2-sulfate and vitamins D2- and D3-sulfate) were detected in human serum (on the order of 0.2–0.6 ng/mL) [38] but not in urine, which raised the hypothesis that these sulfate metabolites serve as 25(OH)D3 reservoirs [36] and are secreted via bile.

Glucuronidation is another conjugation process for vitamin D and its metabolites, and it is mediated by UDP-glucuronosyltransferases (UGT). The most abundant glucuronide of vitamin D in the circulation is 25(OH)D3-glucuronide, which reaches average concentrations of 1.36–2.4 ng/mL [39,40]. On the contrary, one of the major urinary vitamin D3 metabolites in humans is 24,25(OH)2D3-glucuronide (detected in plasma at extremely low concentrations of up to 120 ng/mL), which corresponds to 52.8 ± 19.3 ng/g creatinine [41].

Relatively high serum levels of 25(OH)D-3-sulfate and the ability to be converted to unconjugated 25(OH)D suggest its role as a reservoir of unconjugated forms.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8224373/#B10-nutrients-13-01758

2021 (9)

Circulating Conjugated and Unconjugated Vitamin D Metabolite Measurements by Liquid Chromatography Mass Spectrometry

Results

As a proportion of total circulating vitamin D metabolites, sulfate conjugates (ranging between 18% and 53%) were a higher proportion than glucuronide conjugates (ranging between 2.7% and 11%). The proportion of conjugated 25OHD3 (48 ± 9%) was higher than 25OHD2 conjugates (29.1 ± 10%) across all supplementation groups. Conjugated metabolites correlated with their unconjugated forms for all 4 vitamin D metabolites (r = 0.85 to 0.97).

Conclusion

Sulfated conjugates form a high proportion of circulating vitamin D metabolites, whereas glucuronide conjugates constitute a smaller fraction. Our findings principally in older men highlight the differences in abundance between metabolites and suggest a combination of both conjugated and unconjugated measurements may provide a more accurate assessment of vitamin D status.

Phase II metabolism of vitamin D is less well characterized and comprises largely hepatic conjugation reactions adding a sulfate ester by sulfotransferase (SULT) enzymes or a glucuronide moiety by UDP-glucuronosyltransferase enzymes (6, 7). These conjugation reactions inactivate the vitamin D metabolite and render it more hydrophilic, facilitating renal excretion (8-10). Circulating levels of conjugated vitamin D metabolites have not been well described, although a sulfated form of 25OHD3 (25OHD3-S) in levels that exceed circulating 25OHD3 has been reported (11, 12). This pattern has been confirmed by more recent liquid chromatography tandem mass spectrometry (LC-MS/MS) methods that have also reported a 25OHD3 glucuronide form (25OHD3-G) in circulation at low ng/mL levels (13, 14).

Both sulfate and glucuronide conjugates can undergo deconjugation back to their unconjugated forms by sulfatase and beta-glucuronidase enzymes, respectively. Both deconjugation enzymes have biological roles in local regulation of steroid action within tissues (10, 15)

Application of PVFV-PaS hydrolysis revealed that human serum samples have sulfated forms of vitamin D present at high proportions in circulation relative to the unconjugated forms.

When both beta-glucuronidase and PVFV-PaS are used together with human serum, the proportions of circulating conjugated vitamin D metabolites 25OHD3, 25OHD2, 3-epi-25OHD3, and 24,25(OH)2D3 varies from 29% to 62%. Hence, circulating conjugate forms of vitamin D often match or exceed the more commonly measured unconjugated forms. The proportions of 25OHD3 and 24,25(OH)2D3 as conjugated forms also differ according to vitamin D supplementation status. These findings have clinical relevance for vitamin D measurements as the sulfate form is overlooked by conventional analytical estimation of vitamin D status, a limitation further aggravated by the impact of vitamin D supplementation.

Previous reports have shown that the vitamin D3 metabolites 3-epi-25OHD3 and 24,25(OH)2D3 correlate with increased amounts of circulating 25OHD3 (23). We confirmed these findings, although the proportion of conjugated 3-epi-25OHD3 and 24,25(OH)2D3 to 25OHD3 differed after hydrolysis because of the differences in the proportion of their conjugate forms. Hence measurement of unconjugated metabolites may not reflect the overall differences between the distribution of these conjugated and unconjugated D3 metabolites in circulation. These differences in conjugation status raise further doubt on measurements of unconjugated 25-hydroxyitamin D levels as a sole measure of vitamin D status. In addition to sulfation and glucuronidation, there may be additional esterified vitamin D conjugates (30) that could also contribute to net circulating vitamin D measurements; however, no other additional vitamin D conjugates are known to circulate at significant levels (31). Future studies investigating this by alkaline or esterase hydrolysis would be of interest to appraise any wider spectrum of conjugated metabolites in the assessment of vitamin D status.

Previous studies have reported that 25OHD3-S circulates at similar levels or exceeding the circulating levels of unconjugated 25OHD3 (11, 12, 32). It is also reported that 25OHD3-G is present in circulation at lower ng/mL concentrations (0.5-3 ng/mL) (13, 14). Our study provides a new perspective by measuring simultaneously the concentrations of both unconjugated and conjugated forms of 25OHD3. Our findings indicate that about 50% of 25OHD3 circulates as the sulfate conjugated form, a finding broadly in agreement with a previous study (14). The biological significance for such high circulating levels of conjugated 25OHD3-S is not clear. However, there is minimal renal excretion of the sulfated form as previous studies failed to identify significant amounts of 25OHD3-S in urine (33). This suggests that the sulfated conjugate is unlikely to serve solely as an excretory form of inactivated vitamin D. Alternatively, as 25OHD3-S binds with high affinity to the circulating vitamin D binding protein (DBP) (33), it may form a circulating reservoir available for future deconjugation to 25OHD3 and available to cells for hydroxylation to biologically active 1α,25(OH)2D3 as a paracrine mechanism modulating local vitamin D action in various tissues (33-35). A similar mechanism is reported for steroid sulfate metabolites such as DHEAS and estrone sulfate, which are major circulating forms and are deconjugated by steroid sulfatase for their activation within target tissues (10, 15, 36, 37). This study confirms that 25OHD3-S and other vitamin D sulfate metabolites are substrates for arylsulfatase. Future studies demonstrating 25OHD3-S substrate specificity for human steroid sulfatase would support the hypothesis that 25OHD-S could be deconjugated at local tissue sites due to the ubiquitous presence of steroid sulfatase in human tissues (15, 36).

…it has previously been observed that 25OHD3-S has similar DBP binding affinity as 25OHD3 (33)

While it is possible that 25OHD3-S could be utilized as a storage form for deconjugation at target tissue sites, the role of 24,25(OH)2D3-S in the circulation is less clear and it is generally considered an irreversibly inactive excretory form. Nevertheless, some studies have also described some isolated biological activities for 24,25(OH)2D3 including binding to FAM57B2 stimulating lactosylceramide synthesis which in turn promoted fracture healing in mice (51), along with stimulating growth plate development (52). Although the precise role of circulating 24,25(OH)2D3-S remains unclear, a possible mechanism could involve deconjugation by steroid sulfatase at target sites to undertake these biological actions. A further explanation for the high abundant 24,25(OH)2D3-S could be as a reservoir for deconjugation and further hydroxylation to more biologically active trihydroxyvitamin D3 metabolites; however, this remains to be determined.

In summary, we have demonstrated that the measurements of circulating conjugated fractions of 4 vitamin D metabolites in human serum display a high proportion of circulating conjugated forms of these metabolites, notably the sulfate fraction. These would be overlooked by conventional analytical methods that only measure unconjugated forms. Our findings in this study were in a population-representative cohort of community-dwelling men aged over 70. However, we also observed similarly high levels of the same conjugated vitamin D metabolites in a small cohort of younger male and female samples (Table 2), and further studies are required to determine how well these findings can be extrapolated to different sex and age ranges. The optimized hydrolysis method established in this study will be an important tool in these future studies to understand the precise mechanism of these conjugate metabolites by investigating changes in vitamin D conjugation in health and disease conditions and populations. This could then address the current hypothesis outlined for circulating phase II metabolites (18, 19), such as whether vitamin D conjugation, notably sulfation, represents a process for inactivation and excretion of more hydrophilic inactive metabolites, and/or provides a reservoir storage mechanism for subsequent deconjugation to bioactive forms. Despite previous studies reporting high circulating levels of 25OHD3-S and its potential role in human health, almost all vitamin D analytical measurements intended to evaluate vitamin D status continue to focus on measuring solely unconjugated 25OHD3 and 25OHD2. However, our findings highlight the potential importance of combining these measurements with the measurement of the conjugated forms, especially as the sulfate fraction may constitute a circulating vitamin D binding protein–associated reservoir for bioactive vitamin D. Hence fully assessing vitamin D status may be better reflected by considering the variations in vitamin D conjugation activities that may differ between various metabolites as well as different individuals and disease states.

Our measurements indicate that 25OHD2 conjugates circulate at lower levels than unconjugated 25OHD2 and that D2 supplementation does not appear to alter the proportion of conjugated metabolites. The proportion of 25OHD2 in conjugated form was also lower than in 25OHD3 conjugates for unknown reasons. Both 25OHD3 and 25OHD2 have 3-hydroxyl groups in the alpha configuration (44) and any structural differences occur away from the C-3 sulfation site (33). However, the reduced circulating conjugate forms of 25OHD2 could be explained by the fact that 25OHD2 has a reduced circulating half-life and lower binding affinity for DBP compared with 25OHD3 (44-46). It is also possible that 25OHD2-S could bind with less affinity to DBP than 25OHD3-S as it has previously been observed that 25OHD3-S has similar DBP binding affinity as 25OHD3 (33). Reduced 25OHD2-S binding affinity to DBP could lead to reduced reabsorption and therefore lower circulating levels compared with 25OHD3-S.

Compared with the other vitamin D metabolites, the proportion of 3-epi-25OHD3 circulating as conjugated metabolites was much lower in our samples. This is in broad agreement with a previous study by Yoshimura et al. that reported 3-epi-25OHD3-S in cord blood at lower levels than 3-epi-25OHD3 and also determined that in vitro SULT2A1 sulfation activity for 3-epi-25OHD3 was approximately one-tenth of that for 25OHD3 (20). A possible explanation for reduced SULT2A1 activity for 3-epi-25OHD3 is that sulfation of 25OHD3 predominantly occurs at the C-3 position (33) and the stereochemistry is altered to the beta configuration following C-3 epimerization by 3-epimerase (41, 42), altering enzyme substrate interactions. As 3-epi-25OHD3 and its dihydroxy metabolites are less biologically active than 25OHD3 (43), it remains unclear whether 3-epi-25OHD3-S could be utilized as a storage form or if conjugation of this metabolite is primarily to facilitate excretion as proposed for the other vitamin D sulfate moieties. While the arylsulfatase used in this study appears to show similar levels of activity toward sulfated 3-epi-25OHD3-S and 25OHD3-S, it remains to be determined whether there is a similar level of activity for human steroid sulfatase toward the hydrolysis of these analytes relevant to the proposed the biological significance of the sulfate forms.

We observed that the conjugated forms of 24,25(OH)2D3 are in high abundance in circulation almost exclusively as the sulfate conjugate with minimal glucuronide fraction. The hydroxylation of 25OHD3 to 24,25(OH)2D3 by CYP24A1 is considered to be an inactivation step in the vitamin D3 metabolic pathway (1, 2) with circulating unconjugated 24,25(OH)2D3 correlating strongly with 25OHD3 measurements (23). Further metabolism of 24,25(OH)2D3 can occur to ultimately form calcitroic acid, a urinary and biliary excretory vitamin D product (1, 47). Prior to this 24,25(OH)2D3 is 1-hydroxylated to the more biologically active 1,24,25-trihydroxyvitamin D3 which circulates at low (<25 pg/mL) concentrations (48). The higher abundance of sulfated 24,25(OH)2D3 may be explained by the additional hydroxyl group at the C-24 position that could provide an additional site for sulfation. While it has previously been reported that the C-3 position is the main sulfation site for 25OHD3 (33), the sulfation positions of 24,25(OH)2D3 have not previously been determined; however, the target sites for SULT enzyme activity may be increased if sulfation occurs at both the C-3 and C-24 positions. Excreted 24,25(OH)2D3 has been detected in urine following hydrolysis by beta-glucuronidase (49), so the low circulating amounts of the glucuronide fraction of 24,25(OH)2D3 suggest that it is rapidly excreted into urine following glucuronylation. The binding affinity of 24,25(OH)2D3 to DBP is higher than that of 25OHD3 (46, 50) but the irreversible inactivation by 24 hydroxylation suggests it is unlikely to represent any storage form of vitamin D but rather an excretory product. The high proportion of conjugated 24,25(OH)2D3 conjugates in circulation could be explained by its increased binding to DBP leading to increased kidney reabsorption. While it is possible that 25OHD3-S could be utilized as a storage form for deconjugation at target tissue sites, the role of 24,25(OH)2D3-S in the circulation is less clear and it is generally considered an irreversibly inactive excretory form. Nevertheless, some studies have also described some isolated biological activities for 24,25(OH)2D3 including binding to FAM57B2 stimulating lactosylceramide synthesis which in turn promoted fracture healing in mice (51), along with stimulating growth plate development (52). Although the precise role of circulating 24,25(OH)2D3-S remains unclear, a possible mechanism could involve deconjugation by steroid sulfatase at target sites to undertake these biological actions. A further explanation for the high abundant 24,25(OH)2D3-S could be as a reservoir for deconjugation and further hydroxylation to more biologically active trihydroxyvitamin D3 metabolites; however, this remains to be determined.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9211013/

2022 (2)

Steroid Sulfation in Neurodegenerative Diseases

Like the unconjugated steroids, the sulfated ones modulate various ligand-gated ion channels. Conjugation by sulfotransferases increases steroid water solubility and facilitates steroid transport. Steroid sulfates, having greater half-lives than their unconjugated counterparts, also serve as a steroid stock pool. Sulfotransferases are ubiquitous enzymes providing massive steroid sulfation in adrenal zona reticularis and zona fasciculata.  Steroid sulfatase hydrolyzing the steroid conjugates is exceedingly expressed in placenta but is ubiquitous in low amounts including brain capillaries of BBB which can rapidly hydrolyze the steroid sulfates coming across the BBB from the periphery.

Sulfotransferases

Sulfation takes place by two-step enzymatic reactions; 1) activation of the sulfate group in the form of 3′phosphoadenosine-5′-phosphosulfate (PAPS) by PAPS synthase and 2) transfer of activated sulfate on hydroxyl group of the steroid by SULT (Schiffer et al., 2019). Five cytoplasmic SULTs are known to be involved in steroid metabolism–SULT1A1, SULT1E1, SULT2A1 and 2 isoforms of SULT2B1 (SULT2B1a a SULT2B1b) (Hempel et al., 2000; Fuda et al., 2002; Chang et al., 2004; Gamage et al., 2005). They possess broad substrate specificity; instead of steroid sulfation they can also metabolize phenolic drugs and catecholamines (SULT1A), thyroid hormones (SULT1B) and sterols (SULT2B) (Strott, 2002). Regarding steroids, they can have preferred substrate e.g. SULT1E1 preferentially sulfates estrogens and SULT2A1 most androgens and pregnenolone (reviewed in (Mueller et al., 2015)) and SULT2B1 isoforms stereo-specifically sulfate 3β-hydroxysteroids (e.g., pregnenolone, cholesterol) (Meloche and Falany, 2001; Fuda et al., 2002). SULTs are ubiquitous enzymes with the highest concentrations found in the liver and intestine compared to the kidney and lung (Riches et al., 2009). SULT2A1 is strongly expressed in adrenal zona reticularis, zona fasciculata and the liver. SULT2A1 probably has a dual function: in adrenals it is responsible for massive sulfation of DHEA and pregnenolone and it detoxifies xenobiotics in the liver (Falany and Rohn-Glowacki, 2013).

Regarding the detailed expression of SULTs in brain, SULT1A1 expression was detected in several brain regions (Salman et al., 2009). SULT2A1 was detected exclusively in the thalamus and hypothalamus (Shimizu and Tamura, 2002), it was not detected in other brain regions. These findings are in agreement with the those of Salman et al. who analyzed specimens of prefrontal cortex, hippocampus, and cerebellum (Salman et al., 2011). The results on SULT2B1b expression are ambiguous. No mRNA expression of SULT2B1b in the brain was reported by some authors (Her et al., 1998; Meloche and Falany, 2001), conversely, SULT2B1b mRNA expression was reported in a large number of sections of the human brain by other research groups (Shimizu and Tamura, 2002; Salman et al., 2011). Although SULT2B1a mRNA was detected by Salman et al. (but not by (Shimizu and Tamura, 2002)), no SULT2B1a immunoreactive protein was observed. SULT1E1 expression was not found in human brain in one study (Salman et al., 2011). Taken together, some types of SULTs are apparently available in the brain and therefore may play a crucial role in neurosteroid sulfation and control.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8904904/

2022 (11)

Analysis of the ability of vitamin D3-metabolizing cytochromes P450 to act on vitamin D3 sulfate and 25-hydroxyvitamin D3 3-sulfate

Highlights

CYP27A1 efficiently converts vitamin D3 sulfate to 25(OH)D3 3-sulfate.

CYP11A1 slowly converts vitamin D3 sulfate to 20(OH)D3 3-sulfate.

CYP2R1 does not 25-hydroxylate vitamin D3 sulfate.

Neither CYP27B1 nor CYP24A1 act on 25(OH)D3 3-sulfate.

Sulfation of 25(OH)D3 but not vitamin D3 protects it from oxidation.

Abstract

25-Hydroxyvitamin D3 (25(OH)D3) is present in the human circulation esterified to sulfate with some studies showing that 25(OH)D3 3-sulfate levels are almost as high as unconjugated 25(OH)D3. Vitamin D3 is also present in human serum in the sulfated form as are other metabolites. Our aim was to determine whether sulfated forms of vitamin D3 and vitamin D3 metabolites can be acted on by vitamin D-metabolizing cytochromes P450 (CYPs), one of which (CYP11A1) is known to act on cholesterol sulfate. We used purified, bacterially expressed CYPs to test if they could act on the sulfated forms of their natural substrates. Purified CYP27A1 converted vitamin D3 sulfate to 25(OH)D3 3-sulfate with a catalytic efficiency (kcat/Km) approximately half that for the conversion of vitamin D3 to 25(OH)D3. Similarly, the rate of metabolism of vitamin D3 sulfate was half that of vitamin D3 for CYP27A1 in rat liver mitochondria. CYP2R1 which is also a vitamin D 25-hydroxylase did not act on vitamin D3 sulfate. CYP11A1 was able to convert vitamin D3 sulfate to 20(OH)D3 3-sulfate but at a considerably lower rate than for conversion of vitamin D3 to 20(OH)D3. 25(OH)D3 3-sulfate was not metabolized by the activating enzyme, CYP27B1, nor by the inactivating enzyme, CYP24A1. Thus, we conclude that 25(OH)D3 3-sulfate in the circulation may act as a pool of metabolically inactive vitamin D3 to be released by hydrolysis at times of need whereas vitamin D3 sulfate can be metabolized in a similar manner to free vitamin D3 by CYP27A1 and to a lesser degree by CYP11A1.

Introduction

Vitamin D and its metabolites are present in humans not only in their free form but also as esters. These conjugated forms include sulfates, glucuronides and fatty acid esters [1], [2], [3]. The detection of 25-hydroxyvitamin D3 3-sulfate (25(OH)D3 3-sulfate) in human plasma was first reported in 1985 [4] and it is now well established that it is present in the human circulation at appreciable concentrations, often almost as high as 25(OH)D3 [1], [2], [4], [5], [6], [7], [8], [9]. The plasma of newborns has a higher concentration of 25(OH)D3 3-sulfate than 25(OH)D3 [7], [8], [9]. Recently, Jenkinson et al. [10] reported that the proportion of conjugated 25(OH)D3 was 46–48% of the total in men over the age of 70 both with and without a vitamin D3 supplement. The proportion of sulfate was much higher than the glucuronide with the serum 25(OH)D3 3-sulfate levels averaging 50–55 nM. Some vitamin D3 is also present as the sulfate with a mean serum concentration of 0.7 nM being reported [5]. Conjugated forms of 25-hydroxyvitamin D2, 24,25-dihydroxyvitamin D3 and 3-epi-25-hydroxyvitamin D3 are also present in proportions ranging from 30% to 60% of the total in human serum from men over 70, being highest for 24,25-dihydroxyvitamin D3 [10].

The enzymes responsible for the formation of secosteroid sulfates belong to the sulfotransferases superfamily of enzymes which are responsible for sulfation of steroids, neurotransmitters, drugs and xenobiotic compounds [11]. Sulfating activity on 25(OH)D3 has been found in the liver, kidney, lung and small intestine, and on D3 in the liver. In humans, SULT1A1, SULT2A1, SULT2B1a and SULT2B1b have been shown to sulfate vitamin D3-related compounds [11]. SULT2A1 is believed to be the most important sulfotransferases as it can sulfate 7-DHC, vitamin D3, 25(OH)D3 and 1,25(OH)2D3 [11], [12]. SULT2B1a and SULT2B1b can only use 7-DHC as a substrate and SULT1A1 shows activity only on 1,25(OH)2D3 [11].

Discussion

This study shows that of the three CYPs that can metabolize vitamin D3, both CYP27A1 and CYP11A1 can also metabolize vitamin D3 sulfate, but no appreciable metabolism of the conjugate was observed for CYP2R1. The extent of metabolism of vitamin D3 sulfate by CYP11A1 was lower than for free vitamin D3, more so for the human enzyme than the bovine enzyme.

https://www.sciencedirect.com/science/article/abs/pii/S0960076022001807

2023 (5)

METABOLISM AND PHARMACOKINETICS OF VITAMIN D IN PATIENTS WITH CYSTIC FIBROSIS

Discussion

The present investigation is one of the first to evaluate a comprehensive set of vitamin D metabolites and the pharmacokinetics of 25(OH)D in adults with CF. Only 35% of participants with CF had total 25(OH)D concentrations greater than or equal to the guideline-recommended threshold of 30 ng/mL despite over 50% of them taking vitamin D supplements. Of the metabolites studied, concentrations of total 1α,25(OH)2D, 4β,25(OH)2D3, and 25(OH)D3-S were lower in participants with CF than in healthy controls, even after covariate adjustment. There were no differences in the pharmacokinetics of d6-25(OH)D3 or in the formation of d6-24,25(OH)2D3 between the two groups.

It is possible that low concentrations of 1α,25(OH)2D, 4β,25(OH)2D3, and 25(OH)D3-S are not due to reduced formation, but rather increased elimination.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10524963/

2024 (1)

Analysis of vitamin D3-sulfate and 25-hydroxyvitamin D3-sulfate in breastmilk by LC-MS/MS

Vitamin D metabolites can undergo conjugation with polar substrates to create modified compounds with enhanced solubility and shielded activity [34], [35], [36], [37], [38]. 25-Hydroxyvitamin D3-3-sulfate (25OHD3-S) is notably abundant in serum, at concentrations of 14–38 ng/mL or approximately 40 % of serum 25OHD concentrations [39], [40], [41]. Older reports identified sulfated vitamin D compounds in fresh human milk with concentrations of vitamin D3-3-sulfate (VitD3-S) estimated at 10–24 ng/ml (400–950 IU/L) by thin layer chromatography and HPLC [42], [43], [44]. At the same time, other groups were unsuccessful at measuring the vitamin D-sulfates and concluded that sulfate conjugates were not present in milk [13], [14], [34], [45], [46]. More recently, a method for LC-MS/MS analysis corroborated the notion that unsubstantial amounts of sulfated vitamin D compounds were in breastmilk, but analyzed only the lipid extract [22]. Here, we present an improved method of LC-MS/MS for measuring sulfated vitamin D metabolites in human breastmilk that adequately reflects the enhanced solubility of these modified products and opens the possibility of additional vitamin D metabolites contributing to the milk composition.

Despite the insufficient vitamin D activity of the milk, the serum 25OHD of a nursing infant often correlates with its mother’s serum 25OHD [9,31–33]. This correlation suggests that additional vitamin D metabolites may be transferred to the infant via the mother’s milk

MRM chromatograms revealed an abundance of 25OHD3-S and VitD3-S in the aqueous extract, but little in the organic extract (Figure S1).

In quantification, it was determined that over 90 % of the analytes were collected from the aqueous phase with 3–5 % appearing in the organic extract (Table 5). VitD3-S measurements were more consistent across methods than 25OHD3-S, yielding a mean concentration of 6.1 ± 0.6 ng/mL with a calculated contribution from the breastmilk of 1.4 ± 0.6 ng/mL VitD3-S. 25OHD3-S was less abundant than VitD3-S, ranging from detectable (0.056–0.2 ng/mL 25OHD3-S) to 0.29 ng/mL 25OHD3-S. These findings are approximately 10-times greater than previously described LC-MS/MS methods and correspond to the concentration ranges described for unmodified vitamin D metabolites in human breastmilk [9,13–15,17,18,20–24,26,54]. The contribution of sulfated vitamin D compounds to human breastmilk provides insight for improving our understanding of infant vitamin D nutrition through maternal breastfeeding.

The presence of sulfated vitamin D compounds in the aqueous extract exposes a major caveat in the analysis of sulfated metabolites compared to the unmodified metabolites.

Modification of vitamin D metabolites with the highly polar sulfate increases solubility for enhanced distribution in the mixed matrix of breastmilk. This distribution poses an important consideration for digestion as it becomes plausible that absorption may occur via alternate pathways rather than from chylomicron transport. Additional research on digestion and absorption of these soluble vitamin D conjugates in infants is needed to understand their role in nutrition.

In human breastmilk, VitD3-S is more abundant than 25OHD3-S and can be measured consistently in 250 μL of milk sample. In our assessments we found that (1) method validation of a pooled milk sample contained 1.7 ± 0.23 ng/mL VitD3-S, (2) liquid extractions of a second pooled sample contained 1.4 ± 0.6 ng/mL VitD3-S in the aqueous phase, and (3) cream separations of two individual milk samples contained 0.74 ± 0.33 ng/mL and 0.53 ± 0.33 ng/mL VitD3-S. Taken together, the concentration range of VitD3-S in milk was observed at 0.53–1.7 ng/mL VitD3-S.

25OHD3-S was also present in pooled and individual breastmilk samples at concentrations up to 0.29 ng/mL. Quantification was achievable in experiments that used sample volumes ≥ 500 μL; however, experiments that used 250 μL yielded detectable concentrations with estimated concentrations ranging 0.05–0.2 ng/mL 25OHD3-S. From these experiments, it is recommended that if 25OHD3-S in breastmilk is an analyte of interest, then a sample volume of ≥ 500 μL will provide more reliable quantification.

The abundances of the sulfated metabolites resemble concentrations previously described for unmodified metabolites. Human breastmilk contains < 0.4 ng/mL VitD and 0.1–0.6 ng/mL 25OHD [6,9,13–24,26,54]. In the samples assessed, VitD3-S exceeded the known concentrations for VitD, and 25OHD3-S was near the expected range for 25OHD. These comparisons reveal that sulfated vitamin D metabolites indeed contribute to the total vitamin D content of the breastmilk. While similarly abundant to VitD and 25OHD, the activity and effectiveness of sulfated metabolites in the infant have yet to be determined. In past studies, the role of conjugated vitamin D metabolites were unknown and considered inactive waste products [45,46,55]. Since then, conjugate hydrolysis has been found to enable normal activity [37,38,56–58]. Currently, it is uncertain where, in an infant, sulfatase reactions occur to liberate the vitamin D parent metabolite. If 25OHD-S is cleaved within the digesta, for example, then the resulting 25OHD would be in a free form and may act as a hormonal agonist [58–60]. Internally, the sulfated metabolites may provide a reservoir for vitamin D and 25OHD or target specific cells that express sulfatase enzymes [61–64]. Understanding the metabolic activity of these sulfate conjugates is needed to determine the total nutritive value of sulfated vitamin D metabolites in human breastmilk.

The sample preparation optimized for breastmilk has enabled more robust assessments of VitD3-S and 25OHD3-S by LC-MS/MS than previous efforts. The concentrations of VitD3-S observed in various experiments with pooled and individual milk samples ranged from 0.53 ng/mL to 1.7 ng/mL VitD3-S. 25OHD3-S was less abundant than VitD3-S, ranging from detectable (0.056–0.2 ng/mL 25OHD3-S) to 0.29 ng/mL 25OHD3-S. These findings are approximately 10-times greater than previously described LC-MS/MS methods and correspond to the concentration ranges described for unmodified vitamin D metabolites in human breastmilk [9,13–15,17,18,20–24,26,54]. The contribution of sulfated vitamin D compounds to human breastmilk provides insight for improving our understanding of infant vitamin D nutrition through maternal breastfeeding.

https://www.sciencedirect.com/science/article/pii/S1570023223003641

2024 (9)

Sulfated vitamin D metabolites represent prominent roles in serum and in breastmilk of lactating women

Background

Concentrations of vitamin D (VitD) and 25-hydroxyvitamin D (25OHD) in breastmilk are low despite the essential role of VitD for normal infant bone development, yet additional metabolic forms of vitamin D may be present. This study evaluates the contribution of sulfated vitamin D metabolites, vitamin D3-sulfate (VitD3-S) and 25-hydroxyvitamin D3-sulfate (25OHD3-S) for lactating women and assesses the response to high-dose VitD3 supplementation.

Methods

Serum and breastmilk were measured before and after 28 days with 5000 IU/day VitD3 intake in 20 lactating women. Concentrations of VitD3-S and 25OHD3-S in milk, and 25OHD2, 25OHD3, 25OHD3-S, VitD3 and VitD3-S in serum were determined by mass spectrometry.

Results

Baseline vitamin D status was categorized as sufficient (mean ± SD serum 25OHD3 69 ± 19 nmol/L), and both serum VitD3 and 25OHD3 increased following supplementation (p < 0.001). 25OHD3-S was 91 ± 19 nmol/L in serum and 0.47 ± 0.09 nmol/L in breastmilk. VitD3-S concentrations were 2.92 ± 0.70 nmol/L in serum and 6.4 ± 3.9 nmol/L in breastmilk. Neither sulfated metabolite significantly changed with supplementation in either serum or breastmilk.

Conclusions

Sulfated vitamin D metabolites have prominent roles for women during lactation with 25OHD3-S highly abundant in serum and VitD3-S distinctly abundant in breastmilk. These data support the notion that 25OHD3-S and VitD3-S may have physiological relevance during lactation and nutritional usage for nursing infants.

Sulfate-conjugated forms of vitamin D metabolites were described decades ago in human and bovine milk, but metabolism of these compounds was not well-understood. One study had demonstrated similar effects between VitD and VitD-S on bone repair when fed to vitamin D-deficient adult rats [37]. When fed to nursing rat dams, however, effects from the VitD-S were only observed in the mothers, and not the pups [38,39]. Conjugated metabolites were then categorized as metabolic waste products and dismissed as unimportant [39–42]. Recent investigations of hepatic sulfotransferases as well as bacterial and mammalian sulfatase enzymes demonstrate potential roles for the vitamin D sulfates as sources for VitD and 25OHD or as a mechanism to target specific cells or organs [43–47]. The objective of this study was to assess vitamin D sulfates in serum and breastmilk of lactating women and to evaluate their responses to VitD3 supplementation for the purpose of understanding the magnitude of relevance for sulfated vitamin D metabolites in maternal health and infant nutrition.

3.2 Serum vitamin D sulfates

Serum concentrations of VitD3-S at Day 0 were not different from VitD3 (p = 0.60) and were not different between women who had measurable vs. undetectable levels of VitD3 (p = 0.63, n = 10 per subgroup). Baseline serum concentrations of 25OHD3-S were significantly greater than 25OHD3 (p < 0.001). Neither sulfated analyte changed significantly after high-dose supplementation (Table 2). Variability was consistent for the sulfated metabolites across the cohort with CVs ranging 21–25%, irregardless of timepoint. Relationships of the sulfated metabolites to precursor substrates were assessed to determine feasibility of sulfation and/or hydroxylation pathways (Fig. 1). At baseline, VitD3-S was negatively correlated with VitD3 among participants with measurable VitD3 (n = 10) and not correlated at Day 28 (n = 20). An alternative source for VitD3-S is 7-dehydrocholesterol-sulfate (via sunlight conversion) which was not measured in this study [49]. Trending positive correlations between 25OHD3-S and 25OHD3 were present and similar before and after supplementation, indicating contributions from the sulfation pathway for 25OHD3-S. Hydroxylation was confirmed by positive correlations between VitD3 and 25OHD3; however, no such relationship was observed between VitD3-S and 25OHD3-S. An additional assessment found that VitD3-S had a significant negative correlation with 25OHD3 at the baseline measurement (p = 0.015, r = −0.54), but not following supplementation at Day 28 (p = 0.18, r = −0.31; data not shown).

3.3 Vitamin D sulfates in breastmilk

For all milk samples, VitD3-S was greater than 25OHD3-S (p < 0.001; Table 1), and the two analytes were not correlated (Day 0, p = 0.78, r = −0.07; Day 28, p = 0.99, r = 0.48). As with serum sulfates, high-dose VitD3 supplementation had no effect on the concentrations of VitD3-S or 25OHD3-S in milk. VitD3-S had high variabilities (61% CV Day 0; 81% CV Day 28), whereas 25OHD3-S had low variabilities (19% CV Day 0; 24% CV Day 28). Correlational assessments between the milk and serum analytes were used to identify the serum metabolites contributing to milk (Fig. 2). Concentrations of VitD3-S in milk had no relationship to serum VitD3-S or VitD3, and VitD3-S was 2x greater in breastmilk than in serum. Without significant relationships, the metabolic precursor for VitD3-S in milk cannot be deciphered from these data. Milk 25OHD3-S, on the other hand, was 0.5% of serum 25OHD3-S and had significant positive correlations with serum 25OHD3-S after supplementation and with 25OHD3 at both timepoints. Both transfer of 25OHD3-S across mammary epithelium and local sulfation of 25OHD3 in mammary alveoli are evidently contributing to 25OHD3-S in milk. A summary of the concentrations of all measured metabolites in serum and milk both before and after supplementation are presented in Fig. 3.

3.4 Seasonal variation of vitamin D metabolites

A seasonal decline in vitamin D status was observed for the winter/spring months (January–May) with significantly lower serum 25OHD concentrations than the summer/fall months of June–December (p = 0.004; Supplementary Fig. 1). Serum VitD3-S and 25OHD3-S had no seasonal variation (VitD3-S, p = 0.55; 25OHD3-S, p = 0.36, not shown). Breastmilk VitD3-S had no seasonal variation (p = 0.61), while milk 25OHD3-S was significantly greater in the summer/fall than in the winter/spring (p = 0.039).

3.5 Duration of lactation and vitamin D sulfates

A post-hoc analysis assessed the duration of lactational as a factor affecting the concentrations of sulfated metabolites in the milk. The concentration of VitD3-S in milk had a trending positive correlation with the duration of lactation at baseline (p = 0.050, r = 0.44; Supplementary Fig. 2). Two mothers with low milk VitD3-S concentrations (<2 nmol/L) were both ≤1 month after parturition and both had sufficient serum 25OHD. No relationship was observed between milk 25OHD3-S and duration of lactation.

4 Discussion

The present study characterizes 25OHD3-S and VitD3-S in serum and breastmilk among lactating women and demonstrates how these metabolites do not respond to additional intake of VitD3 by mothers with sufficient vitamin D status. The key findings highlight important contributions of 25OHD3-S in serum during lactation and VitD3-S in breastmilk for infant consumption.

4.1 Serum 25OHD3-S in women during lactation

During lactation, 25OHD3-S was more abundant than 25OHD3 in serum and consistent across the cohort of women. Instances of 25OHD3-S exceeding 25OHD3 have been observed in infants [50,51], whereas 25OHD3-S in adult populations generally corresponds to ∼50% of serum 25OHD and falls within a range of 30–80 nmol/L [52–54]. One study observed a relationship between 25OHD3-S and 25OHD3 and alluded to an apparent seasonal affect [53]. No seasonal effect was observed for serum 25OHD3-S, although women in this cohort remained vitamin D-sufficient during the winter months. Sulfation of 25OHD3, rather than hydroxylation of VitD3-S, appears to be a major pathway contributing to 25OHD3-S in serum. 25OHD3 is a known substrate for the steroid-specific sulfotransferase family 2A member 1 (SULT2A1) enzyme expressed in the liver, adrenal gland, and intestines [43,49]. Hydroxylation of VitD3-S to produce 25OHD3-S is also feasible by CYP27A1 [55], but this pathway was not evident by our correlational assessments. The consistency and abundance of 25OHD3-S demonstrates that it serves a physiological role during lactation and may be a sensitive indicator of nutritional status.

4.2 Serum VitD3-S in women during lactation

VitD3-S in serum was consistent among participants, thereby differing from the highly variable and diet-dependent VitD3 [56]. The metabolic precursor to VitD3-S is presumed to be VitD3 via SULT2A1-mediated sulfation [49]; however, only a negative relationship was observed between VitD3 and VitD3-S that did not continue after high-dose supplementation. Substrate competition may be obscuring this relationship, as VitD3 is the substrate for both VitD3-S and 25OHD3 and an inverse relationship was present between the two products. How nutritional deficiency affects the fate of VitD3 (either by sulfation to VitD3-S or hydroxylation to 25OHD3) remains to be determined and suggests involvement of physiological feedback regulation.

4.3 The accumulated VitD3-S in breastmilk

Decades of research have shown that VitD3 in human milk is less than 1 nmol/L (<16 IU/L) for mothers who follow the recommendation of 600 IU VitD per day [10,22,23,25,57–63]. We found that mothers who were beyond one month of lactation, produced milk with at least 2 nmol/L VitD3-S and mothers who were within one month postpartum secreted less VitD3-S, yet still greater than 1 nmol/L. Our results are 10-fold greater than another investigation that measured the same metabolites by LC-MS/MS during lactation, but the described methods did not account for the enhanced solubility and failed to analyze the aqueous fraction containing >90% of the sulfated metabolites [48,62]. The lower concentrations during early lactation have also been shown for VitD and 25OHD in two investigations, thus differences in colostrum and transition milk composition are expected [22,60]. Perhaps more remarkable were the findings that the VitD3-S concentrations in milk were greater than both serum precursors, VitD3 and VitD3-S, and exhibited no relationship with any of the measured vitamin D metabolites. This distinct abundance of VitD3-S implies that an active mechanism drives VitD3-S accumulation into breastmilk. A proposed model for the pathway of VitD3-S into breastmilk is shown in Fig. 4. The nutritional value of VitD3-S for the infant, however, remains controversial. Investigations on biological activity had assumed that VitD3-S was a biological resource for VitD3, but VitD3-S is ineffective at increasing 25OHD3 and may not follow steroid de-sulfation pathways that apply to dehydroepiandrosterone-sulfate [64,65]. Rather, the sulfated metabolites may utilize bacterial sulfatase from the gut microbiota and/or employ an alternative mechanism for tissue-specific targeting.

4.4 25OHD3-S in breastmilk compares with 25OHD3

The concentrations of 25OHD3-S in milk were consistent across mothers and resembled expected values for 25OHD3. In milk, 25OHD3 ranges from 0.25 to 1.5 nmol/L and corresponds to approximately 1% of serum 25OHD3 [10,22,23,25,57–63,66,67]. Our findings were similar, with 25OHD3-S measurements ranging 0.25–0.69 nmol/L and corresponding to ∼0.5% of serum 25OHD3-S. The positive correlation observed between milk 25OHD3-S and serum 25OHD3, including a seasonal decline in milk 25OHD3-S, suggests that serum 25OHD3 serves as a substrate for local synthesis of milk 25OHD3-S in mammary alveolar cells. The proposed pathways for 25OHD3-S secretion into milk is included in Fig. 4. Again, the physiological role of 25OHD3-S is unknown, although it may exhibit agonistic actions in the colon as has been shown for the glucuronide conjugate of 25OHD3 [45].

4.5 The effects of supplementation on VitD3-S and 25OHD3-S

High-dose VitD3 supplements given to mothers who are already vitamin D-sufficient had no effect on either sulfated vitamin D metabolites in serum or in milk. Even at baseline, no differences were observed between mothers who were taking VitD3-containing supplements (<600 IU per recommended dose) and those who were not. It has previously been established that the unmodified metabolites, 25OHD and VitD, increase from supplementation in serum and proportionally in breastmilk [10,23,25,26,34,57,59,60]. Our study confirmed that supplementation successfully increases both VitD3 and 25OHD3 in serum, but did so without altering sulfated metabolites. The lack of change in sulfated metabolites with supplementation has been indicated in a cross-sectional observational study of adult men (>70 years old) who reported taking or not taking VitD supplements [54]. Men taking supplements had similar 25OHD3-S concentrations to men who did not. The only effect of supplementation on milk analytes that we have reported was a correlation between milk and serum 25OHD3-S that was significant only after supplementation. Thus, increased vitamin D nutrition may simply enable direct transfer of 25OHD3-S from serum to milk. In support, it is possible that DBP is involved in delivering substrate metabolites to the mammary tissue, as both 25OHD3 and 25OHD3-S have similar binding affinities to DBP [43]. Considering the differences in correlations and relative abundances, it is reasonable to postulate that VitD3-S and 25OHD3-S follow separate pathways for secretion in milk. Importantly, the concentrations of both metabolites are independent from the additional VitD3 intake, therefore specific mechanisms are likely involved in preventing excessive accumulation of these metabolites in serum and in milk.

5 Conclusion

We found that both 25OHD3-S and VitD3-S were consistently present in the breastmilk of vitamin D-sufficient mothers. 25OHD3-S was abundant in serum during lactation and VitD3-S was relatively abundant in milk. Supplementation had no effect on the concentrations of either sulfated metabolite in the milk or in the serum. Correlational analyses suggest that 25OHD3-S is produced primarily via sulfation and that both metabolites are under regulation during lactation. We recommend additional investigation of sulfated metabolites to understand their function and role in women's health and in the vitamin D status of infants.

https://www.clinicalnutritionjournal.com/article/S0261-5614(24)00237-1/fulltext