The biological activities of several 24-oxo and 26,23-lactone metabolites of vitamin D were determined in bone organ cultures. The 24-oxo metabolites were significantly more potent bone-resorbing agents than the lactones. 1,25-(OH)2-24-oxo-D3 had 0.18× the bone-resorbing activity of 1,25-(OH)2D3 in fetal rat limb bones and was equipotent with 1,25-(OH)2D3 in neonatal mouse calvaria. In the limb bone system, 1,23,25-(OH)3-24-oxo-D3 had 0.08× the activity of 1,25-(OH)2D3. 1,25-(OH)2D3 and 1,25-(OH)2-24-oxo-D3 had a similar time course of bone-resorbing effects in both bone culture systems. The most potent of the lactones, 1,25S-(OH)2D3-26,23R-lactone, had approximately 0.009× the activity of 1,25-(OH)2D3 and approximately 500 times the activity of the 25S-OH-D3-26,23R-lactone. The 25S and 1,25S lactones were more potent than the 25R and 1,25R isomers. In experiments designed to determine whether either 1,25-(OH)2-24-oxo-D3 or 25R-OH-D3-26,23S-lactone could prevent the bone-resorbing activity of 1,25-(OH)2D3, no inhibitory effects were observed. The results suggest that conversion to the lactones represents a substantial inactivation step, whereas conversion to 24-oxo-derivatives results in less reduction in biological activity.

A sensitive radioimmunoassay system for 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] with an improved extraction procedure has been developed. Following one-step extraction and prepurification of 1,25(OH)2D3 by ‘Extrelut-1’ minicolumns final purification was achieved by high-performance liquid chromatography (HPLC) using a radial compression separation system equipped with a μPorasil cartridge. The HPLC method applied allows the purification of 4 extracts/h. Recovery of 1,25(OH)2[3H]D3 after HPLC was 77 ± 2.6% (mean ± sd, n = 51). Since the recovery of 1,25(OH)2[3H]D3 was very reproducible, addition of labelled steroid to each single serum sample for monitoring recovery was omitted. The sensitivity of the assay was 0.8 pg/tube resulting in a detection limit of 3 ng/1, when 1 ml of serum was extracted. Intra-assay and inter-assay coefficients of variation were 12% and 16.8%, respectively. Serum 1,25(OH)2D3 concentration in 30 normal subjects (mean age: 25 yr) was 55 ± 12 ng/1 (mean ± SD). In 55 elderly patients (mean age: 77 yr) the 1,25(OH)2D3 serum level was 32 ± 12 ng/1 (mean ± SD) and in three patients with chronic renal failure on 1,25(OH)2D3 therapy 146 ± 67 ng/1 (mean ±SD). Patients with chronic renal failure had reduced 1,25(OH)2D3 serum

The binding of the natural and unnatural diastereoisomers 25-hydroxyvitamin D3-26,23-lactone and 1,25 dihydroxyvitamin D3-26,23-lactone to the vitamin D-binding protein (DBP) and 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] chick intestinal receptor have been investigated. Also, the biological activities, under in vivo conditions, of these compounds, in terms of intestinal calcium absorption (ICA) and bone calcium mobilization (BCM), in the chick are reported. The presence of the lactone ring in the C23-C26 position of the seco-steroid side chain increased two to three times the ability of both 25(OH)D3 and 1,25(OH)2D3 to displace 25(OH)[3H]D3 from the D-binding protein; however, the DBP could not distinguish between the various diastereoisomers. In contrast, the unnatural form (23R,25S) of the 25-hydroxy-lactone was found to be 10-fold more potent than the natural form, and the unnatural (23R,25S)1,25(OH)2D3-26,23-lactone three times more potent than the natural 1,25-dihydroxy-lactone in displacing 1,25(OH)2[3H]D3 from its intestinal receptor. While studying the biological activity of these lactone compounds, it was found that the natural form of the 25-hydroxy-lactone increased the intestinal calcium absorption 48 h after injection (16.25 nmol), while bone calcium mobilization was decreased by the same dose of the 25-hydroxy-lactone. The 1,25-dihydroxyvitamin D3-26,23-lactone in both its natural and unnatural forms was found to be active in stimulating ICA and BCM. These results suggest that the 25-hydroxy-lactone has some biological activity in the chick and that 1,25(OH)2D3-26,23-lactone can mediate ICA and BCM biological responses, probably through an interaction with 1,25-(OH)2D3 specific receptors in these target tissues.

Two new vitamin D metabolites were isolated in pure form from separate incubations of homogenates of chick small intestinal mucosa or rat kidney employing either 1 alpha,25-dihydroxyvitamin D3 (28 microM) or 1 alpha,24R,25-trihydroxyvitamin D3 as substrate (0.17-1.3 microM). The newly characterized compounds and the amounts isolated in pure form from separate isolations are, respectively: 1 alpha,25-dihydroxy-24-oxo-vitamin D3 (1,25(OH)2-24-oxo-D3), 147 micrograms from kidney and 4.2 and 40 micrograms from intestine; 1 alpha,23,25-trihydroxy-24-oxo-vitamin D3 (1,23,25(OH)3-24-oxo-D3), 155 micrograms from kidney and 5.9 and 34 micrograms from intestine. Their structures were identified after extensive high pressure liquid chromatography by means of ultraviolet absorption spectrometry, mass spectrometry of the free compounds and their trimethylsilylated derivatives, proton nuclear magnetic resonance spectrometry, specific chemical reduction of the 24-oxo functionality with sodium borohydride, as well as direct comparison with synthetic 1,25(OH)2-24-oxo-D3. These structural assignments for both compounds correct previous determinations which had been proposed (Ohnuma, N., Kruse, J. R., Popjak, G., and Norman, A. W. (1982) J. Biol. Chem. 257, 5097-5102). The activity of the C-24 oxidation pathway used for the production of the 1,25(OH)2-24-oxo-D3 and 1,23,25(OH)3-24-oxo-D3 can be enhanced 10-fold by prior priming of the chicks or rats with a single intravenous dose of 1,25(OH)2D3 (1-12 nmol/100 g body weight); the induction of the enzyme activity is maximal by 3-6 h and returns to basal levels within 12 h. Further, 1,25(OH)2D3, 1,24,25(OH)3D3, and 1,25(OH)2-24-oxo-D3 all were found to be capable of serving as a precursor with chick intestine and rat kidney homogenates of 1,23,25(OH)3-24-oxo-D3. Collectively these results suggest the existence of a C-24 oxidation pathway for metabolism of 1,25(OH)2D3 by the target intestinal mucosa and kidney to 1,23,25(OH)3-24-oxo-D3. The pathway may play an important role in controlling the tissue levels of this hormonally active form of vitamin D3.

Kidney homogenates of rats produced a new metabolite of 25-hydroxyvitamin D3 which has been isolated in pure form after five column chromatographic steps. It was identified as 23,25-dihydroxy-24-oxovitamin D3 by means of ultraviolet and infrared absorption spectrophotometry, mass spectrometry, and proton nuclear magnetic resonance spectrometry. The stereochemistry at the C-23 position is as yet unknown. 25-Hydroxy-24-oxovitamin D3, which also has been isolated in pure form from this system, was found to be the precursor of the new metabolite in vitro. The production of the new metabolite was induced by two different methods: (a) perfusion of the kidneys with 1,25-dihydroxyvitamin D3 contained in the perfusate and (b) injection of 1,25-dihydroxyvitamin D3 in the intact animal. 23,25-Dihydroxy-24-oxovitamin D3 was not biologically active in an assay for intestinal calcium transport and bone calcium mobilization in the vitamin D deficient chick at a dose level of 5.3 nmol. A metabolic pathway is proposed to describe the results; it leads from 25-hydroxyvitamin D3 leads to 24(R),25-dihydroxyvitamin D3 leads to 25-hydroxy-24-oxovitamin D3 leads to 23,25-dihydroxy-24-oxovitamin D3.

Two new metabolites of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], namely 1,25(OH)2-24-oxo-vitamin D3 and 1,23,25(OH)3-24-oxo-vitamin D3, have been prepared in vitro using chick intestinal mucosal homogenates. To investigate the binding of 1,25(OH)2-[23-3H]-24-oxo-D3 and 1,23,25(OH)3-[23-3H]-24-oxo-D3 to the chick intestinal receptor we have isolated both metabolites in radioactive form using an incubation system containing 1,25(OH)2-[23,24-3H))-D3 with a specific radioactivity of 5.6 Ci/mmol. Both metabolites were highly purified by using Sephadex LH-20 chromatography followed by high-pressure liquid chromatography (HPLC). Sucrose density gradient sedimentation analysis showed specific binding of both tritium-labeled metabolites to the chick intestinal cytosol receptor. Experiments were carried out to determine the relative effectiveness of binding to the chick intestinal mucosa receptor for 1,25(OH)2D3. The results are expressed as relative competitive index (RCI), where the RCI is defined as 100 for 1,25(OH)2D3. Whereas the RCI obtained for 1,25(OH)2-24-oxo-D3 was 98 +/- 2 (SE), the RCI for 1,23,25(OH)3-24-oxo-D3 was only 28 +/- 6 (SE). Also, the biological activity of both new metabolites was assessed in vivo in the chick. In our assay for intestinal calcium absorption, 1,25(OH)2-24-oxo-D3 was active at a dose level of 1.63 and 4.88 nmol/bird (at 14 h), whereas 1,23,25(OH)3-24-oxo-D3 showed only weak biological activity in this system. In our assay for bone calcium mobilization, administration of both new metabolites showed modest activity at the 4.88-nmol dose level, which was reduced at the 1.63-nmol dose level. The results indicate that biological activity declines as 1,25(OH)2D3 is metabolized to 1,24R,25(OH)3D3, 1,25(OH)2-24-oxo-D3, and then 1,23,25(OH)3-24-oxo-D3.

AbstractAus dem Hydrindanol‐acetat (I) werden über (II) und (‐III) die Lactone (IV) und (V) hergestellt, die mit dem Dimetliylen‐cyclohexan (VII) zu den Vitamin D3‐Analoga (VI) bzw. (VIII) mit hohen Ausbeuten umgesetzt werden.

1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced alterations in the metabolism of [3H]25-hydroxyvitamin D3 [[3H]25(OH)D3] were studied using isolated perfused kidneys obtained from vitamin D-sufficient rats. Experimental perfused kidneys were exposed invitro to 1,25(OH)2D3 (5×10−8M) for 15 min and then washed with fresh perfusate before the metabolism of [3H]25-(OH)D3 was initiated. Control kidneys from the D-sufficient rats were studied without prior exposure to 1,25(OH)2D3. Tritiated 24R,25-dihydroxyvitamin D3 [24,25(OH)2D3] was the only metabolite produced by both the experimental and control kidneys during the initial two hours of perfusion. However, after two hours of perfusion, the experimental kidneys started producing one major and several minor new tritiated vitamin D metabolites; the production of these compounds then continued until the end of perfusion four hours later. Experimental kidneys, perfused with actinomycin D or cycloheximide failed to produce the new [3H]-metabolites, but continued to produce only 24,25(OH)2D3 similar to control kidneys. Thus the results suggest that 1,25(OH)2D3 acts at the genome to induce the synthesis of enzymes needed for the production of new vitamin D metabolites in obtained kidneys from vitamin D replete rats.

A new metabolite of vitamin D3 has been isolated in pure form from incubations of rat kidney homogenates with 25-hydroxyvitamin D3 [25-OH-D3]. It was identified as 23,25-dihydroxy-24-oxo-vitamin D3 [23,25(OH)2-24-oxo-D3] by means of ultraviolet absorption spectrophotometry and mass spectrometry. Also, 25-OH-D3-26,23-lactone and 24R,25-dihydroxyvitamin D3 were obtained from the same incubation mixtures. The enzyme activity responsible for the conversion of 25-OH-D3 to 23,25(OH)2-24-oxo-D3 was induced by perfusion of the kidneys invitro with 50 nM 1,25-dihydroxyvitamin D3 [1,25(OH)2D3].

The structural requirements for the interaction of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] with an anti-1,25(OH)2D3 antiserum and with the natural cytosolic receptor for 1,25(OH)2D3 isolated from chick intestine have been evaluated quantitatively. The antiserum was raised in a rabbit against a 1,25(OH)2D3-hemisuccinate derivative which was linked to bovine serum albumin at the C-3 position of the steroid. For these cross-reaction studies structural analogs of 1,25(OH)2D3 were used in competitive protein binding assays; their ability to interact with the binding proteins was expressed as relative competitive index (RCI) values where the RCI of 1,25(OH)2D3 is defined to be 100. The results indicate that the 25-hydroxyl group is the most important hydroxyl for the interaction of 1,25(OH)2D3 with this antiserum. The absence of this hydroxyl group decreases the RCI value to 0.2. Lack of the hydroxyl at carbon-3 or carbon-1 decreases the RCI value to 33 or 25, respectively, indicating that the specificity of this antiserum for the A ring is much lower than for the side chain. The high specificity for the side chain is underlined by the fact that insertion of an additional hydroxyl group at C-24 or C-26 of 1,25(OH)2D3 decreases the binding affinity to the antiserum markedly. The chick intestinal mucosal receptor shows a comparable high specificity for the side chain of 1,25(OH)2D3, but an even higher specificity for the A ring in comparison to the antiserum. With the intestinal receptor, the 3-hydroxyl is only 1/ 10th as important as the 1-hydroxyl group and the 25-hydroxyl group for the binding process. Scatchard analysis showed a KD value of 1.7 × 10−10m for the antiserum and 2.3 × 10−10m for the chick intestinal mucosal receptor for the equilibrium binding of 1,25(OH)2D3 at 2 °C. The association rate constant at 2 °C was determined to be 5.8 × 107 M−1 min−1 for the antiserum and 0.55 × 107 M−1 min−1 for the receptor, indicating a 10-fold more rapid association of 1,25(OH)2D3 to the antiserum in comparison to the receptor. Furthermore, the dissociation process was found to be slower for the chick intestinal receptor (dissociation rate constant 3.6 × 10−5 min−1 versus 21.0 × 10−5 min−1).

Since the early 1980s, when pulse oximetry was introduced, this noninvasive method of monitoring the arterial oxygen saturation level in a patient’s blood (SpO2) has become a standard method in the clinical environment because of its simple application and the high value of the information it gives nurses and doctors. It is as common in patient monitoring to measure the oxygen level in the blood as it is to monitor heart activity with the ECG. In some application areas, like anesthesia in a surgical procedure, it is mandatory for doctors to measure this vital parameter. Its importance is obvious considering that a human being cannot survive more than five minutes without oxygen supply to the brain. Before the advent of pulse oximetry, the common practice was to draw blood from patients and analyze the samples at regular intervals—several times a day, or even several times an hour—using large hospital laboratory equipment. These in-vitro analysis instruments were either blood gas analyzers or hemoximeters. Blood gas analyzers determine the partial pressure of oxygen in the blood (pO2) by means of chemical sensors. Hemoximeters work on spectrometric principles and directly measure the ratio of the oxygenated hemoglobin to the total hemoglobin in a sample of blood (SaO2). HP pioneered the first in-vivo technology to measure a patient’s oxygen saturation level without the need of drawing blood samples in 1976 with the HP 47201A eight-wavelength ear oximeter. 1 An earprobe was coupled through a fiber-optic cable to the oximeter mainframe, which contained the light source (a tungsten-iodine lamp and interference filters for wavelength selection) and receivers. This instrument served as a “gold standard” for oximetry for a long time and was even used to verify the accuracy of the first pulse oximeters in clinical studies. The real breakthrough came in the 1980s with a new generation of instruments and sensors that were smaller in size, easier to use, and lower in cost. These new instruments used a slightly different principle from the older, purely empirical multiwavelength technology. Instead of using constant absorbance values at eight different spectral lines measured through the earlobe, the new pulse oximeters made use of the pulsatile component of arterial blood generated by the heartbeat at only two spectral lines. The necessary light was easily generated by two light-emitting diodes (LEDs) with controlled wavelengths. Small LEDs and photodiodes made it possible to mount the optical components directly on the sensor applied to the patient, avoiding the necessity of clumsy fiber-optic bundles.