Single institution followed by national implementation of systematic surgical quality control and feedback for radical prostatectomy: a 20-year journey.

PURPOSE: The demand for objective and outcome-based facts about surgical results after radical prostatectomy (RP) is increasing. Systematic feedback is also essential for each surgeon to improve his/her performance. METHODS: RP outcome data (e.g., pT-stage and margin status) have been registered at Sahlgrenska University Hospital (SUH) since 1988 and patient-related outcome measures (PROM) have been registered since 2001. The National Prostate Cancer Registry (NPCR) has covered all Regions in Sweden since 1998 and includes PROM-data from 2008. Initially PROM was on-paper questionnaires but due since 2018 all PROMs are collected electronically. In 2014 an on-line "dashboard" panel was introduced, showing the results for ten quality-control variables in real-time. Since 2017 all RP data on hospital, regional, and national levels are publicly accessible on-line on "www.npcr.se/RATTEN". RESULTS: The early PROM-data from SUH have been used for internal quality control. As national clinical and PROM-data from the NPCR have been made accessible on-line and in real-time we have incorporated this into our pre-existing protocol. Our data are now internally available as real-time NPCR reports on the individual surgeons' results, as well as ePROM data. We can compare the results of each surgeon internally and to other departments' aggregated data. The public can access data and compare hospital level data on "RATTEN". CONCLUSIONS: The process of quality control of RP locally at SUH, and nationally through the NPCR, has been long but fruitful. The online design, with direct real-time feedback to the institutions that report the data, is essential.

Structural relaxations of phospholipids and water in planar membranes.

We have used dielectric spectroscopy and temperature modulated differential scanning calorimetry (TMDSC) to investigate the structural relaxation processes and phase transitions of water and lipids in multilamellar, planar phospholipids. At low hydration levels we observe the main structural relaxation related to the glass transition of the phospholipids. With increasing water content a more pronounced pretransition, attributed to a gel to ripple phase transition, is observed in the TMDSC data. In the proximity of this pretransition, a distinct change in the temperature dependence or alternatively a bifurcation into two processes is observed in the dielectric data. Around this temperature a crossover in the long-range ionic conductivity across the membranes is also observed, which is one of the key parameters for biological membranes. Thus, the major dynamical changes do not occur at the main, i.e., the gel to liquid structural phase transition, but at a pretransition that occurs roughly 20 K below the main transition.

Renal and hepatic 1 alpha-hydroxylation of 25-hydroxyvitamin D3 in piglets suffering from pseudo vitamin D-deficiency rickets, type I.

The piglets examined suffer from rickets and have symptoms similar to those of classic pseudo vitamin D-deficiency rickets, type I (PVDRI), including plasma concentrations of 1 alpha, 25-dihydroxyvitamin D3 considerably lower than in healthy control piglets. It has been suggested that the rachitic piglets have a defective renal 1 alpha,25-dihydroxyvitamin D3 production. The present study shows that partially purified mitochondrial and microsomal cytochrome P450 from kidney and liver of both rachitic and control animals is able to catalyze 1 alpha-hydroxylation of 25-hydroxyvitamin D3. The renal mitochondrial 1 alpha-hydroxylase activity was higher in the rachitic piglets whereas the renal microsomal 1 alpha-hydroxylase activity was decreased. The immunodetectable levels in kidney of a mitochondrial 1 alpha-hydroxylase (CYP27) and a microsomal 1 alpha-hydroxylase (vitamin D3 25-hydroxylase) were correlated with the 1 alpha-hydroxylase activities. The results suggest that the renal microsomal 1 alpha-hydroxylase is affected by the rachitic condition. It is concluded that the primary genetic defect of systemic 1 alpha,25-dihydroxyvitamin D3 deficiency in the rachitic PVDRI piglets does not reside in a defective function or absence of renal mitochondrial 25-hydroxyvitamin D3 1 alpha-hydroxylase. From this, it may also be concluded that PVDRI in man and pig appear to be two different forms of the disease.

The repressor protein, Bm3R1, mediates an adaptive response to toxic fatty acids in Bacillus megaterium.

Bm3R1 is a helix-turn-helix transcriptional repressor from Bacillus megaterium whose binding to DNA is inhibited by fatty acids and a wide range of compounds that modulate lipid metabolism. The inactivation of Bm3R1/DNA binding activity results in the activation of transcription of the operon encoding a fatty acid hydroxylase, cytochrome P450 102. The metabolic role of this operon is unknown. It is possible that it is involved in the synthesis of modified fatty acids as part of normal cellular metabolism or may represent a protective mechanism by which B. megaterium detoxifies harmful foreign lipids. In this report we demonstrate that polyunsaturated fatty acids (PUFA) activate the transcription of the CYP102 operon. These PUFA are the most potent activators of the CYP102 operon observed to date, and we show that their effects are due to binding directly to Bm3R1. In addition, cultures that have been treated with the CYP102 inducer, nafenopin, are protected against PUFA toxicity. Resistance to PUFA toxicity is also seen in a Bm3R1-deficient strain that constitutively expresses CYP102. The resistant phenotype of this Bm3R1 mutant strain is reversed by specific chemical inactivation of CYP102. These data demonstrate that Bm3R1 can act as a direct sensor of toxic fatty acids and, in addition, provide the first direct evidence of fatty acids binding to a prokaryotic transcription factor.

Cloning, structure, and expression of a cDNA encoding vitamin D3 25-hydroxylase.

The microsomal cytochrome P450 catalyzing the first step in the metabolic activation of vitamin D3 into its hormonal form 1 alpha,25-dihydroxyvitamin D3 has earlier been purified from pig liver. The present communication describes the cloning, structure, and expression of a cDNA encoding pig liver microsomal vitamin D3 25-hydroxylase. DNA sequence analysis of the cDNA revealed a 25-hydroxylase protein of 500 amino acids with a predicted molecular weight of 56,374. The structure of vitamin D3 25-hydroxylase, as deduced by both DNA sequence analysis of the cDNA and protein sequence analysis, shows 70-80% identity with members of the CYP2D family. Transfection of the vitamin D3 25-hydroxylase cDNA into simian COS cells resulted in the synthesis of an enzyme that was recognized by a monoclonal antibody raised against purified vitamin D3 25-hydroxylase and catalyzed 25-hydroxylation in the bioactivation of vitamin D3. Northern blot analysis showed that the mRNA for vitamin D3 25-hydroxylase is found in liver and kidney.

Purification from pig kidney of a microsomal cytochrome P450 catalyzing 1 alpha-hydroxylation of 25-hydroxyvitamin D3.

A cytochrome P450 catalyzing 1 alpha-hydroxylation of 25-hydroxyvitamin D3 was purified from pig kidney microsomes. The enzyme preparation showed one protein band on gel electrophoresis with apparent M(r) of 52,500 and a specific cytochrome P450 content of 10.7 nmol/mg of protein. The 25-hydroxyvitamin D3 1 alpha-hydroxylase copurified with the vitamin D3 25-hydroxylase during purification. A cytochrome P450 catalyzing 1 alpha-hydroxylation was purified also from liver microsomes. The apparently homogeneous enzyme showed the same catalytic properties and apparent M(r) as the kidney enzyme. The results of the present communication demonstrate the presence in kidney of a previously unknown microsomal 1 alpha-hydroxylase in addition to the assumed specific mitochondrial 1 alpha-hydroxylase. The possibility that microsomal 1 alpha-hydroxylation in pig kidney and liver is catalyzed by the previously described porcine microsomal vitamin D 25-hydroxylase(s) is discussed.

Effects on CYP27 mRNA expression in rat kidney and liver by 1 alpha, 25-dihydroxyvitamin D3, a suppressor of renal 25-hydroxyvitamin D3 1 alpha-hydroxylase activity.

The production of 1 alpha,25-dihydroxyvitamin D3 is known to be down regulated by 1 alpha,25-dihydroxyvitamin D3 itself. It was recently shown that liver mitochondrial sterol 27-hydroxylase CYP27, present also in kidney, catalyzes 1 alpha-hydroxylation of 25-hydroxyvitamin D3. Treatment of vitamin D-deficient rats with a single i.v. dose of 1 alpha,25-dihydroxyvitamin D3 resulted in a marked suppression of CYP27 mRNA in kidney. The effects were not as pronounced as for CYP24. Liver CYP27 was not affected to the same extent. The results of the present communication indicate a coordinate regulation of CYP27 mRNA levels and 25-hydroxyvitamin D3 1 alpha-hydroxylase activity by 1 alpha,25-dihydroxyvitamin D3 in rat kidney.

Microsomal 25-hydroxylation of vitamin D2 and vitamin D3 in pig liver.

A microsomal cytochrome P-450 catalysing 25-hydroxylation of vitamin D2 was purified from both male and female pigs to apparent homogeneity and a specific cytochrome P-450 content of 13 and 15.4 nmol x mg of protein-1, respectively. The enzyme also catalysed 25-hydroxylation of vitamin D3. The ratio between the 25-hydroxylase activities towards vitamin D2 and D3 was essentially the same in the different purification steps as well as in the apparently homogeneous enzyme preparation. The two enzyme activities showed the same pH optimum and decreased in parallel upon partial denaturation of the enzyme. Cholecalciferol competitively inhibited 25-hydroxylation of vitamin D2 and vice versa. The non-steroidal cytochrome P-450 inhibitor ketoconazole inhibited both enzyme activities and the Ki values were the same. The cytochrome P-450 showed the same apparent M(r), substrate specificity and N-terminal amino acid sequence as the previously purified vitamin D3 25-hydroxylase from pig liver microsomes. A monoclonal antibody raised against the vitamin D3 25-hydroxylase also recognized the vitamin D2 25-hydroxylase. The antibody immunoprecipitated the 25-hydroxylase activity towards both vitamin D2 and D3 in the purified enzyme. Taken together, the results show that the 25-hydroxylation of vitamin D2 and D3 is catalysed by the same microsomal cytochrome P-450 in pig liver microsomes. The properties of this 25-hydroxylase are discussed in relation to present knowledge concerning previously well-characterized vitamin D3 25-hydroxylases that are not able to catalyse 25-hydroxylation of vitamin D2.

Liver mitochondrial cytochrome P450 CYP27 and recombinant-expressed human CYP27 catalyze 1 alpha-hydroxylation of 25-hydroxyvitamin D3.

A cytochrome P450 catalyzing 1 alpha-hydroxylation of 25-hydroxyvitamin D3 was purified from pig liver mitochondria. It also catalyzed 27-hydroxylation of 25-hydroxyvitamin D3 and 25-hydroxylation of vitamin D3. The ratio between the 1 alpha-, 27-, and 25-hydroxylase activities remained essentially constant during the purification. Substrates for sterol 27-hydroxylase CYP27 inhibited and a monoclonal antibody raised against CYP27 immunoprecipitated the 1 alpha-, 27-, and 25-hydroxylase activities. Apparently homogeneous preparations of CYP27 from pig and rabbit liver mitochondria catalyzed 1 alpha-hydroxylation. Human liver mitochondrial CYP27 was expressed from its cDNA in Escherichia coli. The nucleotide sequence encoding the N terminus of CYP27 was modified in the first eight codons to achieve expression in E. coli. The purified recombinant-expressed CYP27 reconstituted with the electron-transferring system of adrenal mitochondria catalyzed 1 alpha-hydroxylation of 25-hydroxyvitamin D3. Expression of unmodified CYP27 cDNA in simian COS cells confirmed the 1 alpha-hydroxylase activity toward 25-hydroxyvitamin D3.

Purification and characterization of a vitamin D3 25-hydroxylase from pig liver microsomes.

A cytochrome P-450 which catalyses 25-hydroxylation of vitamin D3 has been purified to apparent homogeneity from pig liver microsomes. The specific content of cytochrome P-450 was 12 nmol.mg of protein-1, and the preparation showed a single band with an apparent M(r) of 50,500 upon SDS/PAGE. A monoclonal antibody raised against the vitamin D3 25-hydroxylase reacted strongly with the purified 25-hydroxylating cytochrome P-450 from pig kidney microsomes [Bergman & Postlind (1990) Biochem. J. 270, 345-350]. The liver enzyme showed structural and functional properties very similar to those of the kidney enzyme. The two enzymes differed with respect to only one of the first 16 N-terminal amino acids. The vitamin D3 25-hydroxylase in pig liver microsomes exhibited a turnover and an apparent Km for 25-hydroxylation of vitamin D3 which were of the same order of magnitude as those of a well-characterized male-specific 25-hydroxylating cytochrome P-450 in rat liver microsomes. The two enzymes differed structurally. The pig liver enzyme was, in contrast to the rat liver enzyme, not sex-specific, and did not catalyse 16 alpha-hydroxylation of testosterone. These properties of the 25-hydroxylase in rat liver microsomes have led to questions on the role of microsomal 25-hydroxylation of vitamin D3. It is concluded that studies on microsomal 25-hydroxylation with the rat may be misleading. The results of the present study show that the pig appears to be a representative species for evaluation of vitamin D3 hydroxylases in other mammals, including man.