25-Hydroxycholecalciferol

Metabolites of vitamin D, eg, cholecalciferol (CC), are essential in maintaining the appropriate blood level of Ca ". The active metabolite, 1,25-dihydroxycholecalciferol (1,25-DHCC), is synthesized in two steps. In the fiver, CC is hydroxylated to 25-hydroxycholecalciferol (25-HCC) which, in combination with a globulin carrier, is transported to the kidney where it is converted to 1,25-DHCC. This step, which requites 1-hydroxylase formation, induced by PTH, may be the controlling step in regulating Ca " concentration. The sites of action of 1,25-DHCC are the bones and the intestine. Formation of 1,25-DHCC is limited by an inactivation process, ie, conversion of 25-HCC to 24,25-DHCC, catalyzed by 24-hydroxylase. 

Hydroxy vitamin D pools ia the blood and is transported on DBF to the kidney, where further hydroxylation takes place at C-1 or C-24 ia response to calcium levels. l-Hydroxylation occurs primarily ia the kidney mitochondria and is cataly2ed by a mixed-function monooxygenase with a specific cytochrome P-450 (52,179,180). 1 a- and 24-Hydroxylation of 25-hydroxycholecalciferol has also been shown to take place ia the placenta of pregnant mammals and ia bone cells, as well as ia the epidermis. Low phosphate levels also stimulate 1,25-dihydtoxycholecalciferol production, which ia turn stimulates intestinal calcium as well as phosphoms absorption. It also mobilizes these minerals from bone and decreases their kidney excretion. Together with PTH, calcitriol also stimulates renal reabsorption of the calcium and phosphoms by the proximal tubules (51,141,181—183). 

The metabohtes of vitamin D are usually more toxic than the vitamin because the feedback mechanisms that regulate vitamin D concentrations are circumvented. 25-Hydroxycholecalciferol has a one-hundredfold increase in toxicity over vitamin D when fed to chicks (220) and 1 a,25-dihydroxy vitamin D is several times more toxic than the 25-hydroxy analogue. Vitamin D2 seems to have less toxicity than vitamin D, a circumstance which is beheved to be caused by the more efficient elimination of 25-hydroxy and the 1 a,25-dihydroxy vitamin D2 from the animals. Estimated safe upper dietary levels are given in Table 11. 

Chemical Name 9,10-Secocholesta-5,7,10(l9)-triene-3,25-diol Common Name 25-Hydroxyvitamin D3 25-Hydroxycholecalciferol Structural Formula ... 

A solution of 20 mg of 25-hydroxychoiecaiciferoi hydrate, prepared as described above, in 20 mi of methyiene chioride is dried with 200 mg of anhydrous sodium suifate. The solution is fiitered and the fiitrate is evaporated to yield 25-hydroxycholecalciferol essentially anhydrous as an amorphous oil. 

The following are recent reviews on the molecular and physical properties of this liver enzyme which converts cholecalciferol (vitamin D3) to 25-hydroxycholecalciferol. 

Mitochondrial system The function of the mitochondrial cyto chrome P450 monooxygenase system is to participate in the hydroxylation of steroids, a process that makes these hydropho bic compounds more water soluble. For example, in the steroid hormone-producing tissues, such as the placenta, ovaries, testes, and adrenal cortex, it is used to hydroxylate intermediates in the conversion of cholesterol to steroid hormones. The liver uses this system in bile acid synthesis (see p. 222), and the kidney uses it to hydroxylate vitamin 25-hydroxycholecalciferol (vitamin D, see p. 384) to its biologically active 1,25-hydroxylated form. 

Formation of 1,25-diOH D3 Vitamins D2 and D3 are not biologically active, but are converted in vivo to the active form of the D vitamin by two sequential hydroxylation reactions (Figure 28.23). The first hydroxylation occurs at the 25-position, and is catalyzed by a specific hydroxylase in the liver. The product of the reaction, 25-hydroxycholecalciferol (25-OH D3), is the predominant form of vitamin D in the plasma and the major storage form of the vitamin. 25-OH D3 is further hydroxylated at the one position by a specific 25-hydroxycholecalciferol 1 -hydroxylase found primarily in the kidney, resulting in the formation of 1,25-dihydroxycholecalciferol j (1,25-diOH D3). [Note This hydroxylase, as well as the iver 25-hydroxylase, employ cytochrome P450, molecular oxygen, and NADPH.]... 

Regulation of 25-hydroxycholecalciferol 1-hydroxylase 1,25-diOH D3 is the most potent vitamin D metabolite. Its formation is tightly i regulated by the level of plasma phosphate and calcium ions (Figure 28.24). 25-Hydroxycholecalciferol1 -hydroxylase activity is I increased directly by low plasma phosphate or indirectly by bw I plasma calcium, which triggers the release of parathyroid hormone I... 

Which one of the following statements concerning vitamin D is correct A. Chronic renal failure requires the oral administra tion of 1,25-dihydroxycholecalciferol. B. It is required in the diet of individuals exposed to sunlight. C. 25-Hydroxycholecalciferol is the active form of the vitamin. D. Vitamin D opposes the effect of parathyroid hor mone. E. A deficiency in vitamin D results in an increased secretion of calcitonin. Correct answer = A. Renal failure results in the decreased ability to form the active form of the vitamin, which must be supplied. The vitamin is not required in individuals exposed to sunlight. 1,25-dihydroxycholecalciferol is the active form of the vitamin. Vitamin D and parathyroid hormone both increase serum calcium. A deficiency of vitamin D decreases the secretion of calcitonin. 

KT Koshy, AL VanDerSlik. High-performance liquid chromatographic method for the determination of 25-hydroxycholecalciferol in the bovine liver, kidney, and muscle. J Agric Food Chem 25 1246-1249, 1977. 

P Mattila, V Piironen, E Uusi-Rauva, P Koivistoinen. Determination of 25-hydroxycholecalciferol in egg yolk by HPLC. J Food Comp Anal 6 250-255, 1993. 

KT Koshy, AL VanDerSlik. 25-Hydroxycholecalciferol in cow milk as determined by high-performance liquid chromatography. J Agric Food Chem 27 650-652, 1979. 

Schreurs WH, van Rijn HJ, van den Berg H. Serum 25-hydroxycholecalciferol levels in women using oral contraceptives. Contraception 1981 23(4) 399 t06. 

The combination of rifampicin and isoniazid reduces serum concentrations of 25-hydroxy cholecalciferol. Rifampicin acts by induction of an enzyme that promotes conversion of 25-hydroxycholecalciferol to an inactive metabolite, and isoniazid acts by inhibiting 25-hydroxyla-tion and 1-hydroxylation (SEDA-14, 258). Children or pregnant women with tuberculosis have increased calcium requirements independent of rifampicin administration... 

Vitamin D-binding protein and its associated vitamin are lost in nephrotic urine. Biochemical abnormalities in nephrotic patients (children and adults) include hypocalcemia, both total (protein-bound) and ionized hypocalciuria, reduced intestinal calcium absorption and negative calcium balance reduced plasma 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol and, surprisingly, also 1,25-dihydroxycholecalciferol and blunted response to parathormon (PTH) administration and increased PTH levels. Clinically, both osteomalacia and hyperparathyroidism have been described in nephrotic patients, more commonly in children than in adults, but bone biopsies are commonly normal, and clinically significant bone disease is very rare in nephrotic subjects. There is, however, evidence that patients with renal failure accompanied by nephrotic range proteinuria may be particularly prone to develop renal osteodystrophy. 

The fractions which crystallize on trituration with aqueous methanol are combined and recrystallized twice from aqueous methanol to give 25-hydroxycholecalciferol hydrate yield 120 mg, MP 81°-83°C (sinters 75°C). 

Cholecalciferol is hydroxylated at three positions in the carbon skeleton, 1, 24, and 25. In the liver, cholecalciferol is hydroxylated to 25-hydroxycholecalciferol. Further hydroxylation reactions occur in the kidney, resulting in the formation of three new metabolites. These are 1,25-dihydroxycholecalciferol 24,25-dihydroxycholecalciferol and 1,24,25-trihydroxycholecalciferol. 1,25-Dihydroxy- and 1,24,25-trihydroxycholecalciferol are active hormones involved in calcium uptake from the intestine. 

Both dietary and endogenously synthesized vitamin D undergo 25-hy-droxylation in the liver to yield calcidiol (25-hydroxycholecalciferol), which is the main circulating form of the vitamin. This undergoes 1 -hydroxylation in the kidney to produce the active hormone calcitriol (1,25-dihydroxy-cholecalciferol) or 24-hydroxylation in the kidney and other tissues to yield 24-hydroxycalcidiol (24,25-dihydroxycholecalciferol). 

Disposition in the Body. Well absorbed after oral administration and subject to enterohepatic circulation decreased absorption may occur in subjects with impaired liver and biliary function. Metabolised by hydroxylation to active metabolites. The major metabolite is 25-hydroxycholecalciferol which is formed in the liver. This is further metabolised by la- or 24-hydroxylation in the kidneys. Most of a dose is excreted in the bile and eliminated in the faeces about 25% of a dose is excreted as conjugates. Unchanged cholecalciferol does not appear to be excreted in the urine. 

Blood Concentration. Normal serum concentrations of 25-hydroxycholecalciferol are about 0.015 to 0.040 pg/ml, but there are considerable intersubject and seasonal variations. Half-life. Plasma half-life, about 1 to 2 days after intravenous administration a terminal elimination half-life of about 18 days has been reported. 

It has been suggested that vitamin D3 (378) is metabolized into a more polar substance before stimulating calcium transport to the intestine. The principal metabolite from the blood, produced by the liver, has been found to be 25-hydroxycholecalciferol (379), whereas the trihydroxy-derivative (380) is the principal metabolite from the intestine. Autoxidation of cholesterol via hydroperoxide intermediates afforded a variety of hydroxylated cholesterol derivatives and products of side-chain degradation. ... 

Osteomalacia, a very rare manifest form of hepatic osteopathy, causes pain mainly in the muscles, but less so in the bones. Biochemically, AP is markedly increased there is a deficiency of calcium, phosphate and 25-hydroxycholecalciferol. Radiologic diagnosis shows signs of Looser-Milkman s syndrome coarsening of cancellous bone structure, narrowing of the compacta in the tubular bones, reduced density of the skeletal system, and band-shaped zones of decalcification. 

The product of the reaction 25-hydroxycholecalciferol or 25 -OH D3 is the predominant form of vitamin D in the plasma and is the major form in which vitamin D is stored. 

Bone mineral density has been measured in 59 patients and 55 age- and sex-matched controls (118). Bone mineral density in the lumbar spine (L2-4) and femurs was lower in the patients, significantly so in the former case. This reduction depended on the duration of therapy. Excretion of pyridinoline cross-links was markedly increased and 25-hydroxycholecalciferol and 1,25-dihydroxycholecalci-ferol were significantly reduced. The proliferation rate of human osteoblast-hke cells was increased by phenytoin in low doses. 

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