Joanne O. Whitney

Use of Sep-pak cartridges for urinary steroid extraction: evaluation of the method for use prior to gas chromatographic analysis.

A method is described for the rapid and quantitative extraction of free and conjugated steroids from urine using Sep-pak C18 cartridges. The method was evaluated by determining the efficiency of recovery of (1) radiolabeled steroid glucuronides, (2) radiolabeled steroids freed by enzymatic hydrolysis, (3) steroid sulphates, (4) selected reference neutral free steroids of varied structure and polarity, and (5) oestrogens. In all cases the cartridges gave results equal to or better than those obtained by solvent or Amberlite XAD-2 extraction methods. Each urine extraction could be completed in 2-3 minutes and no further purification of extracts was required prior to derivatisation and gas chromatographic analysis.

A Simple Liquid Chromatographic Method for Quantitative Extraction of Hydrophobic Compounds from Aqueous Solutions

Abstract We are reporting a rapid, high-capacity liquid chromatographic method for quantitative extraction and concentration of hydrophobic compounds from biological fluids and aqueous solutions. Samples are injected into commerically-available cartridges (Sep-Pak C18R) containing a microparticulate, reversed phase packing which retains hydrophobic compounds. Inorganic salts and organic hydrophilic contaminants are removed with a water wash. Hydrophobic compounds are eluted quantitatively with minimal volumes (∼5 ml) of organic solvents. As demonstrated with radiolabeled taurocholate, thin-layer chromatography, enzymatic fluorimetry and capillary gas chromatography, complete recovery of bile salts from large volumes of urine, serum, amniotic fluid and hydrolysis reaction mixtures was achieved at flow rates up to 20 ml/min. A single cartridge concentrated approximately 50 mg of either taurocholate or the more polar bile salt, taurolithocholate sulfate. The technique is simple and applicable to the isolatio...

In vitro synthesis of vitamin D-3 by cultured human keratinocytes and fibroblasts: action spectrum and effect of AY-9944.

With delineation of the photochemical events occurring in the skin after ultraviolet exposure, there has been increased interest in the skins role in the vitamin D-3-endocrine system. We provide here in vitro conditions for the generation of both labelled (from [3H]acetate) and unlabelled vitamin D-3 in cultures of human keratinocytes and fibroblasts. Sterol precursors and photoproducts in irradiated and non-irradiated cultures are identified by co-chromatography, ultraviolet absorbance spectra, thermal conversion characteristics of previtamin D-3 and mass spectrometry. Because the conversion of 7-dehydrocholesterol to cholesterol is more efficient in vitro than in vivo, the specific delta 7 inhibitor, AY-9944, was added in non-toxic doses to modulate 7-dehydrocholesterol content. Both cell types were equally capable of generating photoproducts, depending on the amount of 7-dehydrocholesterol present. The 290 +/- 5 and 295 nm filters were much more efficient than the 305 nm filter for generating previtamin D-3 and vitamin D-3 in fibroblasts. In contrast, the 305 nm filter was as efficient as the 290 +/- 5 and 295 nm filters in keratinocytes, where it yielded previtamin D-3, with much less lumisterol and tachysterol than appeared with the shorter-wavelength filters. The amount of lumisterol and tachysterol versus previtamin D-3 formed in both cell types was dependent on the total energy applied, with lower energies (less then 1 J/cm2) favoring previtamin D-3 over the other photoproducts. The use of cultured cells provides a system whereby the regulation of vitamin D-3 synthesis by extracutaneous factors can be studied in a homogeneous setting.

Vitamin D3 production by cultured human keratinocytes and fibroblasts

We have demonstrated that monolayers of human cultured newborn foreskin keratinocytes and fibroblasts elaborate vitamin D3 following exposure to UV-B. This in vitro system provides a new means to study those factors (hormones, ions, vitamin D3 metabolites, etc.) that regulate the production of vitamin D3 by human skin cells. Vitamin D3 production was enhanced greatly by using cells that were pre-treated with AY-9944, a non-toxic drug that inhibits cholesterologenesis while elevating cellular levels of 7-dehydrocholesterol, the sterol precursor of vitamin D3. The pre-D3 formed within viable, irradiated cells is transformed to D3 within a matter of hours at 37 degrees C, and keratinocytes proved to be more proficient sources of the vitamin and its metabolites than corresponding skin fibroblasts.

Urinary metabolites of vitamin D3

Abstract Relatively little is known regarding the identity of the terminal metabolites of vitamin D 3 , yet measurement of these compounds would give additional useful data on the normal and pathophysioogy of vitamin D in man. We have therefore investigated the metabolism of [26.27- 3 H]-25-hydroxy vitamin D 3 administered orally to a human subject. A relatively minor proportion of radioactivity was excreted in urine during the first 7 days of collection and faecal excretion was not determined. The state of conjugation of the urinary metabolites was determined by fractionation of Amberlite XAD-2 organic extracts by Sephadex DEAR LH-20 chromatography before and after hydrolysis. Of the [ 3 H]-radioactivity recovered, 15% was excreted as neutral compounds, 60% as glucuronides of neutral steroids. 8%, as acids released by glucuronidase hydrolysis and 5% as monosulphates. In an additional experiment, an attempt has been made to identify (by GC/MS) metabolites in urine of a patient with hypophosphalemic vitamin D resistant rickets (VDRR) following administration of an oral dose of 5 mg vitamin D 3 . The primary metabolites of vitamin D 3 (e.g. 25-hydroxy vitamin D 3 ; 24,25-dihydroxy vitamin D 3 ) were not detected, but five components gave mass spectra indicative of vitamin D 3 structure. All five components had 25-hydroxyl groups; one almost certainly had, in addition, a 24-hydroxyl and one a 26-hydroxyl. The possibility that they were hydroxylated sterols can be tentatively excluded by the fact that these compounds were not found in the urine of the same patient during a period when he was off vitamin D therapy.

Mitochondrial granulation in the proximal renal tubule in uremia.

Mitochondria contain electron-dense particles, partly composed of an amorphous form of calcium phosphate. We have used electron microscopy from percutaneous renal biopsy material to analyze mitochondrial granulation in the proximal renal tubule of nonuremic and uremic children. Based on a technique of cutting mitochondria from ten electron micrographs per biopsy, counting the granules in each mitochondrion and weighing the paper, we found that mitochondria of nonuremic children averaged 23.7 +/- 1.2 granules/g paper while uremic children had only 11.8 +/- 1.1 granules/g. The number of granules per gram was unrelated to the serum calcium phosphate solubility product. A significant decrease in calcium granulation in uremia can also be produced experimentally in rats. Control rats averaged 14.7 +/- 1.5 granules/g, while rats made uremic by partial nephrectomy had 6.0 +/- 0.7 granules/g. Treatment of uremic rats with a pharmacological dose of vitamin D restored granulation to normal within 24 h. The significant decrease in calcium phosphate granulation in the renal proximal tubule in uremic children and in experimental animals is probably related to the documented loss of 1 alpha-hydroxylation of vitamin D in uremia.

Quantitation of Osteolytic Phytosteryl Acetates in Human Serum

Summary A gas-chromatographic method for the quantitation of phytosteryl acetates in human serum at the nanogram level is described. Cholesteryl acetate is utilized as an internal standard added to the serum before extraction. Using this method, serum phytosteryl acetate levels of breast cancer patients are not significantly different from those of other subjects.

Disparate Effects of Vitamin D Treatment upon Mitochondrial Granulation in Proximal and Distal Renal Tubule

Abstract The distribution of calcium phosphate granules in mitochondria of proximal and distal renal tubules of nonuremic and uremic children was analyzed by electron microscopy of material obtained by percutaneous kidney biopsy. Although distal tubule had fewer granules/ mitochondrion than proximal tubule, uremia induced a significant drop (50%) in both, related to an increase in mitochondria containing 0 granules and a decrease in mitochondria with 2+ granules. The decrease observed in uremic children was reproduced experimentally by partial nephrectomy in rats. Uremia resulted in a 58% decrease of calcium phosphate granules in rat proximal tubule while a smaller but significant decrease (36%) occurred in distal tubule. Vitamin D deficiency in rats was associated with greatly decreased granulation in proximal tubule (80%) whereas distal tubule was less severely affected (36%). Supplementation of vitamin D to uremic rats restored mitochondrial granulation to normal in proximal tubule in 24 hr, but had no effect in distal tubule since the number of granules/mitochondrion, 0.5 ±0.1, remained statistically similar to that of untreated animals. Granulation in both proximal and distal tubule of uremic rats was unaffected by parathyroid hormone administration. Since restoration of granulation occurred only in proximal tubule, the defect in uremia which can be overcome by vitamin D treatment appears localized at the level of the proximal tubular cell membrane, indicating an action of vitamin D on calcium and/or phosphorus translocation into the proximal tubule.

401 TRANSPLACENTALLY INDUCED LIVER INJURY DUE TO LITHOCHOLATE: CANALICULAR Na, K-ACTIVATED ATPase AND MEMBRANE MICROVISCOSITY IN NEWBORNS

Administration of lithocholate (L) to pregnant rats has been shown to induce inflammatory lesions in liver of newborn offspring, progressing to intrahepatic biliary hypoplasia and chronic cholestasis (Gastroenterology 73:1214, 1977). The site of action and mechanism of L toxicity in utero was investigated in newborn rats delivered by dams maintained on standard chow (S) or S+2.5% L throughout pregnancy. Newborns were sacrificed at 1,2, and 5 days after birth, plasma collected, and liver removed for histological examination and isolation of canaliculi-enriched plasma membranes. Membrane Na, K-activated ATPase activity was assayed, and surface microviscosity (mV) determined by fluorescence polarization. Plasma L concentration was 2-3 μmol/L in treated, and was undetectable in untreated newborns. Hepatic changes in L-treated newborns were periportal inflammation, formation of pseudoducts and giant cells. ATPase activity in membranes from L-treated newborns was reduced by 40% compared with normals (2.58±0.30 vs 4.14±0.45 μmol P/hr/mg protein). Canalicular mV (in poises) was increased by 30% in L-treated newborns compared with normals (5.65±0.30 vs 3.99±0.18; p<0.01). Conclusion: Exposure to L in utero is associated with changes in canalicular membrane function and physical properties known to interfere with bile secretion, and may thus initiate a self-perpetuating cholestatic disorder after birth.