Calcium balance during calcitriol and paricalcitol administration in healthy humans.

OBJECTIVE: Secondary hyperparathyroidism in hemodialysis patients requires optimal correction of vitamin D deficiency with active vitamin D and analogues. It has been postulated that new vitamin D analogues, i.e. paricalcitol, efficiently suppress parathyroid hormone serum levels (PTH), but do not increase intestinal calcium absorption as much as calcitriol. The effects of calcitriol and paricalcitol on calcium balance can best be characterized under standardized conditions in healthy individuals with normal renal function, because the urinary calcium excretion at steady state corresponds to the net calcium absorption in the gut. METHODS: In a randomized, double-blind, placebo-controlled, 3-way crossover Phase I study in 13 healthy individuals we investigated the changes compared to placebo in PTH and urinary calcium excretion during 6-day treatment periods with paricalcitol (1.5 microg/day) and calcitriol (0.5 microg/day). RESULTS: 24-hour urinary calcium excretion was stable during 6 days of placebo administration. Neither paricalcitol nor calcitriol significantly changed calcium excretion. Urinary creatinine, magnesium and phosphate excretion also remained unchanged over the study periods irrespective of the treatment. However, calcitriol was shown to be effective in reducing iPTH levels during 6 days of treatment (mean reduction 4.03+/-0.69 pmol/l), whereas paricalcitol had no effect. CONCLUSION: Using a dosing ratio of 1:3 for calcitriol:paricalcitol, i.e. the same conversion factor used previously in studies on hemodialysis patients, only calcitriol was able to reduce iPTH levels in healthy individuals. Low-dose calcitriol reduced iPTH levels without raising calcium absorption and without including any hypercalcemia.

Evaluation of sulfonated aluminum phthalocyanines for use in photochemotherapy. Cellular uptake studies.

Cellular uptake of aluminum phthalocyanine sulfonated to different degree was studied by means of fluorescence measurements and HPLC chromatography. These results were correlated to the lipophilic property of each drug measured as the distribution of the drug between a lipophilic phase (Triton X-114) and an aqueous phase. All the sulfonated aluminum phthalocyanines were taken up into cells to a higher extent than porphyrins of a similar lipophilicity. The cellular uptake of monosulfonated aluminum phthalocyanine was 10-fold higher than the cellular uptake of tetrasulfonated aluminum phthalocyanine and at least 50% higher than tetra(3-hydroxy-phenyl)porphin which is so far the porphyrin shown to be taken up into cells to the highest extent.

Osteodystrophy in the millennium.

Despite three decades of intensive research on the derangements of calcium phosphate metabolism of renal failure, several unresolved issues are still with us at the turn of the millennium: poor control of hyperphosphatemia, relative inefficacy of active vitamin D to prevent progressive parathyroid hyperplasia, and persistence of bone disease despite lowering of parathyroid hormone (PTH) and administration of active vitamin D. Although predictions are problematic, it is not unreasonable to hope that, barring unforeseen side effects, calcimimetics will prove to be valuable for suppressing or even preventing hyperparathyroidism, thus potentially replacing, at least in part, active vitamin D. There is also reason to hope that more effective phosphate binders with fewer side effects will become available and that controlled studies will provide a rationale for the administration of estrogens to dialyzed women. As regards understanding the pathological mechanisms, one can anticipate that the disturbances leading to autonomous growth of parathyroid cells will be elucidated and the signals involved in osteoclast/osteoblast differentiation pathways and osteoclast/osteoblast coupling will be clarified, with obvious impact on patient management.

Photobleaching of mono-L-aspartyl chlorin e6 (NPe6): a candidate sensitizer for the photodynamic therapy of tumors.

Most sensitizers used for the photodynamic therapy (PDT) of tumors photobleach on illumination. Thus, it is of interest to examine the photobleaching behavior of new sensitizers proposed for use in PDT. This report surveys the quantum yields and kinetics of the photobleaching of mono-L-aspartyl chlorin e6(NPe6), a hydrophilic chlorin that has many of the photoproperties desirable in a sensitizer for clinical PDT. It is a very effective sensitizer for the PDT of several types of model tumors in animals and is now in Phase I clinical trials. The quantum yield of NPe6 photobleaching in pH 7.4 phosphate buffer in air was 8.2 x 10(-4); this is greater than the yields for typical porphyrin photosensitizers. For example, the yields for hematoporphyrin and uroporphyrin are 4.7 x 10(-5) and 2.8 x 10(-5), respectively. The yield decreased significantly in organic solvents of low dielectric constant. The Sn derivative of NPe6 was more light stable than NPe6 (yield = 5.7 x 10(-6), while the Zn derivative was more sensitive (yield = 1.9 x 10(-2). Oxygen appeared to be necessary for the photobleaching of NPe6; however, bleaching was not inhibited by 100 mM azide, an efficient quencher of singlet oxygen. The photooxidizable substrates cysteine, dithiothreitol and furfuryl alcohol increased the quantum yield of photobleaching two- to four-fold, while the electron acceptor, metronidazole, increased it almost six-fold. Photobleaching yields for several other chlorins were also measured.

Evaluation of sulfonated aluminum phthalocyanines for use in photochemotherapy. A study on the relative efficiencies of photoinactivation.

The cellular photosensitivity caused by aluminum phthalocyanines sulfonated to different degrees (AlPcSn) has been investigated. The phototoxic effect increased with decreasing number of sulfonate groups on the macrocycle, with the exception of AlPcS1 which was less phototoxic than AlPcS2 but more phototoxic than AlPcS3 and AlPcS4. The tendency of the AlPcSns to aggregate in our cellular system increased with increasing lipophilicity of the sensitizers. The aggregates had little or no photosensitizing activity. The low efficiency of cell inactivation caused by AlPcS1 can be explained by the highly aggregated state of this sensitizer in the cells. AlPcS2 and AlPcS3 induced a lower degree of cell inactivation per fluorescing quantum and per quantum absorbed by monomeric species than did AlPcS2 and AlPcS1. AlPcS4 and AlPcS3 are therefore suggested to be in different intracellular locations than AlPcS2 and AlPcS1.

Intracellular localization of sulfonated meso-tetraphenylporphines in a human carcinoma cell line.

The intracellular localization of meso-tetraphenylporphines sulfonated to different degrees (TPPSn), in a human cervix carcinoma cell line (NHIK 3025), was studied by fluorescence microscopy and fluorescence spectroscopy. After an 18 h incubation, TPPS4, TPPS2a and TPPS2o were localized in extranuclear granules. Studies of cells stained with both TPPS4, and acridine orange, which is known to fluoresce red in lysosomes, indicated that these granules were lysosomes. In addition, a fraction of the cellbound TPPS4, TPPS2a and TPPS2o seems to be associated with the plasma membrane. Fluorescence quenching studies of cells doublestained with acridine orange and TPPS4 indicated that TPPS4 is also localized in the nucleus and in the extralysosomal cytoplasm. The intracellular location of TPPS1 differed from that of the other TPPSns studied: In 6 out of 9 experiments fluorescing extranuclear granules were found. A diffuse fluorescence extending from the perinuclear area was also observed.

Action spectra of phthalocyanines with respect to photosensitization of cells.

Human carcinoma cells (NHIK 3025 cells) and Chinese hamster cells (V79 cells) were incubated with AlPcS1, AlPcS2 and AlPcS4, phthalocyanines with different lipophilicity but with similar photochemical properties when in monomeric solutions. The absorption- and fluorescence spectra of the dyes in the cells were recorded as well as their action spectra with respect to sensitizing cells to photoinactivation. These spectra show that under the present conditions AlPcS1 is strongly aggregated in both cell lines; AlPcS2 is aggregated in V79 cells but much less so in NHIK 3025 cells. A main finding is that the shapes of the action spectra are similar to that of the fluorescence excitation spectra, but not to the absorption spectra, indicating that the photosensitizing effects of the dyes are mainly due to their monomeric fraction in the cells. AlPcS2 and AlPcS4 localize intracellularly mainly in lysosomes while AlPcS1 was found to be more diffusely distributed in cells. As measured per quantum of fluorescence emitted, AlPcS1 and AlPcS2 are more efficient sensitizers than AlPcS4. The difference in efficiency between AlPcS2 and AlPcS4 is supposedly due to a different localization pattern on the suborganelle level.

Cellular uptake and relative efficiency in cell inactivation by photoactivated sulfonated meso-tetraphenylporphines.

The cellular uptake, relative fluorescence quantum yields and photosensitizing efficiencies of meso-tetraphenylporphines sulfonated to different degrees (TPPSn) have been investigated using the human carcinoma cell line NHIK 3025. The efficiencies of these dyes in photoinactivation of cells were highly dependent on the number of sulfonate groups on the derivatives. These differences in phototoxicity were primarily due to different abilities to be taken up by cells, but were also dependent upon the cellular localization of the dyes. TPPS1 and TPPS2a were more efficiently taken up by the cells than TPPS2o and TPPS4. Plasma membrane associated TPPS4 was less efficient in cell inactivation per quantum of fluorescence emitted than intracellularly located dye. This was also to some extent the case for TPPS1 but not for TPPS2a and TPPS2o. The results presented here indicate that TPPS2a and TPPS1 are the most promising of the TPPSns for possible future use in photodynamic therapy.

Light induced relocalization of sulfonated meso-tetraphenylporphines in NHIK 3025 cells and effects of dose fractionation.

Human cervix carcinoma cells of the line NHIK 3025 were incubated for 18 h with sulfonated meso-tetraphenylporphines (TPPSn where n = 1, 2a, 2o or 4) followed by 1 h in sensitizer-free medium and then exposed to light. The fluorescing fraction of TPPS4, TPPS2o and TPPS2a has recently been shown to be located intracellularly in extracellular granules which are intracellularly localized in a similar pattern as acridine orange-stained granules, assumed to be endosomes and lysosomes (Berg, K., A. Western, J. Bommer and J. Moan. Photochem. Photobiol. 52, 481-487). Light exposure induced a relocalization of TPPS4 from its granular pattern to mainly the nuclear area while TPPS2o and TPPS2a relocalized mainly to cytoplasmic areas. After the light-induced relocalization TPPS4 became less efficient in sensitizing photoinactivation of cells as measured per fluorescing cellbound TPPS4 molecules while TPPS2a and TPPS2o became more efficient. These changes were independent of the extracellular concentration of TPPSn applied to the cells, except for cells incubated with 75 micrograms/mL TPPS4. These cells became more sensitive to light after a light exposure inactivating 20% of the cells. This increased photosensitivity seems to be related to a 2-2.5 fold increase in the amount of fluorescing cellbound TPPS4 induced by the first light exposure.

Strategies for selective cancer photochemotherapy: antibody-targeted and selective carcinoma cell photolysis.

A principle objective in chemotherapy is the development of modalities capable of selectively destroying malignant cells while sparing normal tissues. One new approach to selective photochemotherapy, antibody-targeted photolysis (ATPL) uses photosensitizers (PS) coupled to monoclonal antibodies (MAbs) which bind to cell surface antigens on malignant cells. Selective destruction of human T leukemia cells (HBP-ALL) was accomplished by coupling the efficient PS chlorin e(6) to an anti-T cell MAb using dextran carriers. Conjugates with chlorin: MAb ratios of 30:1 retained > 85% MA b binding activity, and had a quantum yield for singlet oxygen production of 0.7 +/- 0.1, the same as that of free chlorin e(6). Cell killing was dependent on the doses of both MAb-PS and 630-670 nm light and occurred only in target cell populations which bound the MAb. On the order of 10(10) singlet oxygen molecules were necessary to kill a cell. A second approach to specific photochemotherapy, selective carcinoma cell photolysis (SCCP), relies on preferential accumulation of certain cationic PS by carcinoma cell mitochondria. We have evaluated several classes of cationic dyes, and in the case of N,N'-bis-(2-ethyl-1,3-dioxolane)-kryptocyanine (EDKC) and some of its analogs, have demonstrated highly selective killing of human squamous cell, bladder and colon carcinoma cells in vitro. In isolated mitochondria, EDKC uptake and fluorescence depended on membrane potential, and the dye specifically photosensitized damage to Complex I in the electron transport chain. N,N'-bis-(2-ethyl-1,3-dioxolane)-kryptocyanine and some of its analogs accumulated within subcutaneous xenografts of human tumors in nude mice with tumor:skin ratios > 8. Photoirradiation caused significant inhibition of tumor growth, without cutaneous phototoxicity.

Cellular inhibition of microtubule assembly by photoactivated sulphonated meso-tetraphenylporphines.

This work relates to studies on modes of phototoxicity by sulphonated mesotetraphenylporphines on cultured cells. Toxicity appears to be related to inhibition of microtubule function. Treatment of human cervix carcinoma cells of the line NHIK 3025 incubated for 18 h with meso-tetraphenylporphine sulphonates (TPPSn where n = 2a, 2o or 4) and exposed to light, inhibits multiplication for the first hours after light exposure, a significant fraction of the cells accumulating in mitosis. The maximal number of cells in mitosis after treatment (approximately 20%) is dependent on the fluence but is similar for all three photosensitizers. For the first hours after treatment the mitotic cells were always mainly in metaphase; mainly seen as c-metaphases and three-group metaphases. During this time anaphase and telophase cells were absent or greatly reduced in number. Indirect immunofluorescence staining of beta-tubulin showed that the spindle apparatus of mitotic cells was perturbed in all cases. Results are presented which indicate that photoactivation of TPPSn located on the plasma membrane destroys microtubules in interphase cells and leads to arrest of the cells in mitosis. The localization of the dye which sensitizes the photoinduced perturbation of microtubules is further discussed.

Sexual behaviour of hemodialyzed patients.

18 married men on home dialysis and 10 of their wives were interviewed (structured interview) to evaluate sexual adjustment. The frequency of intercourse prior to the onset of their disease was reported to be 3.4/week by the patients and 2.4/week by their wives. During maintenance hemodialysis, frequency was reported to be 1.7/week by the patients and 1.0/week by their wives. If libido prior to the disease was rated 100%, the patients reported their libido at the time of the interview as 66+/-34%; only 5 rated their libido as less than 50% of their libido prior to the onset of their disease. After the start of dialysis, considerable time was required before a stable state of sexual activity was reached (13.9+/-7.4 months), presumably reflecting the time required for adaptation to the procedure, loss of anxiety and restoration of physical well-being. Except in older patients, there was good agreement between the feeling of physical fitness as reported by the patients and sexual performance as reported by both the patient and his spouse.

No evidence for endotoxin transfer across high flux polysulfone membranes.

Recently, there has been some concern that high-flux membranes may expose dialysis patients to the risk of endotoxin transfer secondary to backfiltration within the dialyzer. To evaluate the safety of high-flux polysulfone dialyzers, we examined in an in vitro recirculation system whether lipopolysaccharides (LPS) and lipid A respectively penetrate from the dialysate to the blood compartment and vice versa using a F-60 hemofilter (Fresenius AG). For the detection of endotoxin, a sensitive, kinetic limulus amebocyte lysate (LAL) microtiter test was used. It can be concluded that LPS and lipid A do not pass from either side through the filter system used when saline was recirculated for more than 10 h on both sides of the membrane.

Acquired renal cysts in uremic patients--in vivo demonstration by computed tomography.

The development of renal cysts appears to be a common feature of terminal renal failure in patients with diffuse renal parenchymal disease. In the present investigation, the kidneys of 13 patients with terminal renal failure but not receiving dialysis, of 14 patients on maintenance hemodialysis and of 4 patients after renal transplantation (patients' own kidneys) were studied by computed tomography. Cystic lesions in the contracted renal parenchyma could be demonstrated by computed tomography in 7/13 non-dialyzed patients with terminal renal failure, in 11/14 patients on maintenance hemodialysis as well as in 3/4 transplanted patients (patients' own kidneys). Both solitary cysts (10/21 patients) and multiple cysts (11/21 patients) were observed. The size varied from 0.5 cm (barely detectable) to 3 cm in diameter. Such cysts could also be demonstrated at autopsy. Possible clinical complications include spontaneous retroperitoneal hemorrhage, macrohematuria, matrix stone formation and formation of benign or malignant papilloma. The present study shows that computed tomography allows the detection of acquired renal cysts in uremic patients in vivo. The cysts appear prior to dialysis, seem to increase in frequency during dialysis and do not disappear after transplantation. The lesions can be distinguished from multicystic or polycystic disease.

Long-term comparison of a calcium-free phosphate binder and calcium carbonate--phosphorus metabolism and cardiovascular calcification.

BACKGROUND: Calcium carbonate used as a phosphate binder may contribute to cardiovascular calcification. Long-term comparisons of sevelamer, a non-calcium polymeric phosphate binder, and calcium carbonate (CC) are lacking. METHODS: 114 adult hemodialysis patients were randomly assigned to open label sevelamer or CC for 52 weeks. Study efficacy endpoints included changes in serum phosphorus, calcium, calcium-phosphorus product, and lipids. In addition, initial and sequential electron beam computerized tomography scans were performed to assess cardiovascular calcification status and change during follow-up. Safety endpoints were serum biochemistry, blood cell counts and adverse events. RESULTS: Patients receiving sevelamer had a similar reduction in serum phosphorus as patients receiving CC (sevelamer -0.58 +/- 0.68 mmol/l, CC -0.52 +/- 0.50 mmol/l; p = 0.62). Reductions in calcium-phosphorus product were not significantly different (sevelamer -1.4 +/- 1.7 mmol2/l2, CC -0.9 +/- 1.2 mmol2/l2; p = 0.12). CC produced significantly more hypercalcemia (> 2.8 mmol/l in 0% sevelamer and 19% CC patients, p < 0.01) and suppressed intact parathyroid hormone below 150 pg/ml in the majority of patients. Sevelamer patients experienced significant (p < 0.01) reductions in total (-1.2 +/- 0.9 mmol/l, -24%) and LDL cholesterol (-1.2 +/- 0.9 mmol/l, -30%). CC patients had significant increases in coronary artery (median +34%, p < 0.01) and aortic calcification (median +32%, p < 0.01) that were not observed in sevelamer-treated patients. Patients on sevelamer required more grams of binder (sevelamer 5.9 g vs. CC 3.9 g) and experienced more dyspepsia than patients on calcium carbonate. CONCLUSIONS: Sevelamer is an effective phosphate binder that unlike calcium carbonate is not associated with progressive cardiovascular calcification in hemodialysis patients.

Photosensitizing properties of mono-L-aspartyl chlorin e6 (NPe6): a candidate sensitizer for the photodynamic therapy of tumors.

There is a large amount of interest in chlorins as photosensitizers for the photodynamic therapy of tumors because of their strong absorption in the red, where light penetration into mammalian tissues is efficient. Mono-L-aspartyl chlorin e6 (NPe6), in phosphate buffer of pH 7.4, had absorption peaks at 400 and 654 nm with molar absorption coefficients of 180,000 and 40,000 M-1 cm-1 respectively. In buffer, the NPe6 triplet had a peak at 440 nm and a lifetime under argon of approximately 300 microseconds. The triplet was efficiently quenched by ground state oxygen (kQ = 1.9 x 10(9) M-1 s-1) with the formation of singlet oxygen, as identified by its near infrared luminescence. The quantum yield of singlet oxygen production was 0.77. A number of substrates were efficiently photo-oxidized by NPe6, including furfuryl alcohol, cysteine, histidine, tryptophan and human serum albumin. These reactions were efficiently inhibited by azide (which did not quench NPe6 triplets), indicating that they are probably mediated by singlet oxygen. Thus, NPe6 has a desirable array of photoproperties for a sensitizer to be used in the clinical photodynamic therapy of tumors.

HBV DNA and other hepatitis B virus markers in sera from long-term hemodialysis and kidney transplant patients.

Sera from 191 long-term hemodialysis patients and from 115 renal transplant patients were studied for the presence of HBsAg and other HBV markers. In 19.1% of the hemodialysis patients and 28.7% of the transplant patients, the sera were positive for HBeAg.HBV DNA in sera was detected by molecular hybridization. HBV DNA was present in the sera of 15 out of 16 hemodialysis patients positive for HBeAg, and in one hemodialysis patient positive for anti-HBe. All renal transplant patients positive for HBeAg were also positive for HBV DNA. Twelve transplant patients were positive for HBV DNA, but negative for both HBeAg and anti-HBe. Determination of HBV DNA was the most sensitive marker of infectivity in patients with end-stage renal disease, and in renal transplant patients.

Elimination of Staphylococcus aureus in hemodialysis patients.

Staphylococcus aureus infection and its complications are of great concern in the care of hemodialysis patients. Nasal contamination with S. aureus seems to be the main source of cutaneous contamination. The decontamination and recontamination of the skin of hemodialysis patients after using mupirocin nasal ointment was followed in a placebo control study. After 10 days of therapy with mupirocin nasal ointment, 25 of 33 (73%) patients were free of nasal S. aureus contamination in the nares (control subjects 2 of 21, 10%). At the same time, the prevalence of positive skin cultures for S. aureus decreased from 30 of 33 (90%) to 11 of 33 (33%) patients. However, during the ensuing 130 days, 14 of 25 (58%) patients with negative nasal cultures became recontaminated, while the skin became recontaminated in 11 of 22 (50%) patients. In 10 of 14 S. aureus recontaminated patients the original S. aureus lysotype was documented by specific phage reaction. Four of fourteen patients had a new S. aureus lysotype. Mupirocin nasal ointment eradicated S. aureus transiently in 75% of the patients but continuously in only 11 of 33 (30%) patients.

Arteriosclerosis in dialysis patients.

Arteriosclerosis is a constant problem in long-term hemodialysis patients. Computer tomography of the abdominal aorta allows a well-defined and reproducible evaluation of aortosclerosis. In the cross-sectional study, aortosclerosis was significantly accelerated in 84 chronic hemodialysis patients and was comparable to the results found in 20-year older control patients without renal disease. The increase of aortosclerosis correlated significantly with age of the patient, smoking, and duration of dialysis therapy. Furthermore, increased VLDL cholesterol and decreased HDL cholesterol seem to enhance aortosclerosis in our dialysis patients. In the longitudinal study (two CT scans with a time interval of 87 +/- 62.7 months) in 36 dialysis patients, progressed aortosclerosis correlated significantly with the long duration of hypertriglyceridemia, VLDL cholesterol, uric acid, and calcium phosphate products. Progression of aortosclerosis was reduced in parathyroidectomized patients. The study suggests that premature aortosclerosis is found in dialysis patients. In addition to the common risk factor of aortosclerosis disturbed calcium phosphate and parathyroid hormone metabolism seem to enhance aortosclerosis in patients under maintenance hemodialysis.