Scavenging interactions between the arrow tooth eel Synaphobranchus kaupii and the Portuguese dogfish Centroscymnus coelolepis.

A scavenging interaction between the arrow tooth eel Synaphobranchus kaupii and the Portuguese dogfish Centroscymnus coelolepis, both ubiquitous components of fish assemblages at bathyal depths, was observed. Using a baited camera between 1297 and 2453 m in the eastern Atlantic Ocean continental slope, it was shown that despite consistently rapid arrival times of S. kaupii (<5 min), their feeding bouts (indicated by acute peak in numbers) did not take place until shortly after C. coelolepis arrived and removed the exterior surface of the bait (skipjack tuna Katsuwonus pelamis carcass). Change in the numbers of S. kaupii was hence dependent on the arrival of a more powerful scavenger throughout the study site, and at the deeper stations where the population of C. coelolepis declined, S. kaupii was observed to be present but waited for >2 h before feeding, thus contradicting conventional scavenging assumptions in the presence of a food fall.

Liparid and macrourid fishes of the hadal zone: in situ observations of activity and feeding behaviour.

Using baited camera landers, the first images of living fishes were recorded in the hadal zone (6000-11000 m) in the Pacific Ocean. The widespread abyssal macrourid Coryphaenoides yaquinae was observed at a new depth record of approximately 7000 m in the Japan Trench. Two endemic species of liparid were observed at similar depths: Pseudoliparis amblystomopsis in the Japan Trench and Notoliparis kermadecensis in the Kermadec Trench. From these observations, we have documented swimming and feeding behaviour of these species and derived the first estimates of hadal fish abundance. The liparids intercepted bait within 100-200 min but were observed to preferentially feed on scavenging amphipods. Notoliparis kermadecensis act as top predators in the hadal food web, exhibiting up to nine suction-feeding events per minute. Both species showed distinctive swimming gaits: P. amblystomopsis (mean length 22.5 cm) displayed a mean tail-beat frequency of 0.47 Hz and mean caudal:pectoral frequency ratio of 0.76, whereas N. kermadecensis (mean length 31.5 cm) displayed respective values of 1.04 and 2.08 Hz. Despite living at extreme depths, these endemic liparids exhibit similar activity levels compared with shallow-water liparids.

Limitations of randomized clinical trials for evaluating emerging operations: the case of lung volume reduction surgery.

Although unanswered questions remain, scores of observational studies and several small randomized clinical trials (RCTs) indicate that lung volume reduction surgery (LVRS) offers safe and effective palliation for a relatively well defined subset of patients with advanced emphysema. Nonetheless, Medicare and other insurers stopped reimbursement for the procedure. Subsequently, two multicenter RCTs on LVRS, the National Emphysema Treatment Trial (NETT) and the Overholt-BlueCross Emphysema Surgery Trial (OBEST), were launched with the stipulation that the procedure would not be paid for outside these trials. Thus access to LVRS has been denied to patients who could benefit but do not wish to participate in an RCT. Emerging operations, unlike new drugs or devices, pass through evolutionary changes and frequently fail to produce data that meet the scientific rigor required by randomized studies. In such a setting, the observational approach is more appropriate. Indeed, almost all operations in the present surgical armamentarium have been evaluated and have evolved through observational studies without the use of RCTs. By the time new operations are standardized and qualify for RCTs, benefits for certain patients may be demonstrated and randomization could involve unacceptable health hazards. Patients from this population should be offered the choice between participating in RCTs and having the operation outside the study. Imposition of financial restrictions that bars access to a therapy with known benefit is a questionable practice.

Mice deficient in methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid deposition.

Hyperhomocysteinemia, a risk factor for cardiovascular disease, is caused by nutritional and/or genetic disruptions in homocysteine metabolism. The most common genetic cause of hyperhomocysteinemia is the 677C-->T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene. This variant, with mild enzymatic deficiency, is associated with an increased risk for neural tube defects and pregnancy complications and with a decreased risk for colon cancer and leukemia. Although many studies have reported that this variant is also a risk factor for vascular disease, this area of investigation is still controversial. Severe MTHFR deficiency results in homocystinuria, an inborn error of metabolism with neurological and vascular complications. To investigate the in vivo pathogenetic mechanisms of MTHFR deficiency, we generated mice with a knockout of MTHFR: Plasma total homocysteine levels in heterozygous and homozygous knockout mice are 1.6- and 10-fold higher than those in wild-type littermates, respectively. Both heterozygous and homozygous knockouts have either significantly decreased S-adenosylmethionine levels or significantly increased S-adenosylhomocysteine levels, or both, with global DNA hypomethylation. The heterozygous knockout mice appear normal, whereas the homozygotes are smaller and show developmental retardation with cerebellar pathology. Abnormal lipid deposition in the proximal portion of the aorta was observed in older heterozygotes and homozygotes, alluding to an atherogenic effect of hyperhomocysteinemia in these mice.

Treatment of hyperhomocysteinemia in hemodialysis patients and renal transplant recipients.

BACKGROUND: Hyperhomocysteinemia, a putative atherothrombotic risk factor, is observed in at least 85% of patients undergoing maintenance hemodialysis (HD), as well as 65 to 70% of renal transplant recipients (RTRs). The hyperhomocysteinemia regularly found in HD patients is largely refractory to combined oral vitamin B supplementation featuring supraphysiological doses of folic acid (FA). Relative to their HD counterparts, the hyperhomocysteinemia of RTRs appears to be considerably less refractory to treatment with high-dose FA-based vitamin B supplementation regimens, although controlled comparison data are lacking. We evaluated whether improved total homocysteine (tHcy)-lowering efficacy could be achieved in chronic HD patients with a high-dose L-5-methyltetrahydrofolate (MTHF)-based regimen, as suggested by recent uncontrolled findings, and compared the relative responsiveness of RTRs and HD patients with equivalent baseline tHcy levels, to 12 weeks of tHcy lowering with combined folate-based vitamin B treatment. METHODS: First, we blocked randomized 50 chronic, stable HD patients based on their screening predialysis tHcy levels, sex, and dialysis center into two groups of 25 subjects treated for 12 weeks with oral FA at 15 mg/day, or an equimolar amount (17 mg/day) of oral MTHF. All 50 subjects also received 50 mg/day of oral vitamin B6 and 1.0 mg/day of oral vitamin B12. RESULTS: The mean percentage (%) reductions (+/- 95% confidence intervals) in predialysis tHcy were not significantly different [MTHF 17.0% (12.0 to 22.0%), FA 14.8% (9.6 to 20.1%), P = 0.444 by matched analysis of covariance adjusted for pretreatment tHcy]. Final on-treatment values (mean with 95% confidence interval) were: MTHF, 20.0 micromol/L (18.8 to 21.2); and FA, 19.5 micromol/L (18.3 to 20.7). Moreover, neither treatment resulted in "normalization" of tHcy levels (that is, final on-treatment values <12 micromol/L) among a significantly different or clinically meaningful number of patients [MTHF, 2 out of 25 (8%); FA, 0 out of 25 (0%); Fisher's exact test of between groups difference, P = 0.490]. Second, we compared the relative responsiveness of (N = 10) RTRs and (N = 39) HD patients with equivalent baseline tHcy levels (RTR range of 14.2 to 23.6 micromol/L, and HD range of 14.4 to 24.9 micromol/L) to 12 weeks of tHcy-lowering treatment. The RTRs received 2.4 mg/day of FA, 50.0 mg/day of vitamin B6, and 0.4 mg/day of vitamin B12, while the HD patients received 15 mg/day of FA or an equimolar amount (17 mg/day) of the reduced folate, MTHF, in addition to 50.0 mg/day of vitamin B6 and 1.0 mg/day of vitamin B12. The mean percentage (%) reductions (+/- 95% confidence interval) in tHcy were as follows: RTR 28.1% (16.2 to 40.0%); HD 12.1% (6.6 to 17.7%, P = 0.027 for comparison of between groups differences by analysis of covariance adjusted for baseline tHcy levels). Moreover, 5 out of 10 (50.0%) of the RTR versus only 2 out of 39 (5.1%) of the HD patients had final on-treatment tHcy levels <12 micromol/L (P = 0.002 for comparison of between groups differences by Fisher's exact test). CONCLUSIONS: First, in comparison to high-dose FA, high-dose oral MTHF-based supplementation does not afford improved tHcy-lowering efficacy among HD patients. The preponderance of HD patients (that is,> 90%) exhibits mild hyperhomocysteinemia refractory to treatment with either regimen. This treatment refractoriness is not related to defects in folate absorption or circulating plasma and tissue distribution. Second, relative to RTR with comparable baseline tHcy levels, the mild hyperhomocysteinemia of maintenance HD patients is much more refractory to tHcy-lowering vitamin B treatment regimens featuring supraphysiological amounts of FA or the reduced folate MTHF. Accordingly, RTRs are a preferable target population for controlled clinical trials testing the hypothesis that tHcy-lowering vitamin B intervention may reduce arteriosclerotic cardiovascular disease event rates in patients with chronic renal disease.

Conversion of 5-formyltetrahydrofolic acid to 5-methyltetrahydrofolic acid is unimpaired in folate-adequate persons homozygous for the C677T mutation in the methylenetetrahydrofolate reductase gene.

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the synthesis of 5-methyltetrahydrofolic acid (5-CH(3)-H(4) folic acid), the methyl donor for the formation of methionine from homocysteine. A common C677T transition in the MTHFR gene results in a variant with a lower specific activity and a greater sensitivity to heat than the normal enzyme, as measured in vitro. This study was undertaken to determine the capacity of homozygotes for the MTHFR C677T transition to convert 5-formyltetrahydrofolic acid (5-HCO-H(4) folic acid) to 5-CH(3)-H(4) folic acid, a process that requires the action of MTHFR. Six subjects homozygous for the C677T transition (T/T) and 6 subjects with wild-type MTHFR (C/C) were given a 5-mg oral dose of (6R:,S:)-5-HCO-H(4) folic acid. Plasma and urine were analyzed for 5-CH(3)-H(4) folic acid concentrations using affinity/HPLC coupled with fluorescence or UV detection. The mean areas under the curves created by the rise and fall of plasma 5-CH(3)-H(4) folic acid after the oral dose did not differ between the two genotypes, 424.5 +/- 140.3 (T/T) vs. 424.1+/- 202.4 h.nmol/L (C/C). There also was no significant difference in the mean cumulative 7-h urinary excretion of 5-CH(3)-H(4) folic acid between the T/T (2.5 +/- 1.4 micromol) and C/C (1.9 +/- 1.0 micromol) genotypes. Under the conditions employed, the conversion of oral 5-HCO-H(4) folic acid to 5-CH(3)-H(4) folic acid is not impaired in persons with the T/T MTHFR genotype. Possible reasons for these findings are discussed.

Controlled comparison of L-5-methyltetrahydrofolate versus folic acid for the treatment of hyperhomocysteinemia in hemodialysis patients.

BACKGROUND: The hyperhomocysteinemia regularly found in hemodialysis patients is largely refractory to combined oral B-vitamin supplementation featuring supraphysiological doses of folic acid. We evaluated whether a high-dose L-5-methyltetrahydrofolate-based regimen provided improved total homocysteine (tHcy)-lowering efficacy in chronic hemodialysis patients. METHODS AND RESULTS: We block-randomized 50 chronic, stable hemodialysis patients on the basis of their screening predialysis tHcy levels, sex, and dialysis center into 2 groups of 25 subjects treated for 12 weeks with oral folic acid at 15 mg/d (FA group) or an equimolar amount (17 mg/d) of oral L-5-methyltetrahydrofolate (MTHF group). All 50 subjects also received 50 mg/d of oral vitamin B(6) and 1.0 mg/d of oral vitamin B(12). The mean percent reductions (+/-95% CIs) in predialysis tHcy were not significantly different: MTHF, 17.0% (12.0% to 22.0%); FA, 14.8% (9.6% to 20.1%); P=0.444 by matched ANCOVA adjusted for pretreatment tHcy. Final on-treatment values (mean with 95% CI) were MTHF, 20.0 micromol/L (18.8 to 21.2 micromol/L); FA, 19.5 micromol/L (18.3 to 20.7 micromol/L). Moreover, neither treatment resulted in "normalization" of tHcy levels (ie, final on-treatment values <12 micromol/L) among a significantly different or clinically meaningful number of patients: MTHF, 2 of 25 (8%); FA, 0 of 25 (0%); Fisher's exact test of between-groups difference, P=0.490. CONCLUSIONS: Relative to high-dose folic acid, high-dose oral L-5-methyltetrahydrofolate-based supplementation does not afford improved tHcy-lowering efficacy in hemodialysis patients. The preponderance of hemodialysis patients (ie, >90%) exhibit mild hyperhomocysteinemia refractory to treatment with either regimen. This treatment refractoriness is not related to defects in folate absorption or circulating plasma and tissue distribution.

Effect of chronic alcohol consumption on total plasma homocysteine level in rats.

BACKGROUND: Chronic alcoholism in humans is associated with the development of hyperhomocysteinemia, the mechanism of which remains unclear. Among the causes of hyperhomocysteinemia is depletion of folate, vitamin B12, or vitamin B6. Population-based studies indicate that folate is the strongest vitamin determinant of hyperhomocysteinemia and, in most settings, folate supplementation effectively lowers elevated homocysteine levels. However, it is not clear whether folate deficiency is the cause of alcohol-related hyperhomocysteinemia. METHODS: In the present study, 10 male Sprague Dawley rats were fed ethanol-containing Lieber-DeCarli diets with 13 mg of folic acid per kilogram of diet. This represents a folate intake more than 20 times the basal requirement. Ethanol represented 36% of total energy, which yielded a concentration of 6.2% (vol/vol). The same number of rats were pair-fed with isocaloric control diets that contained an identical level of folate in which ethanol was entirely replaced by maltodextrin. RESULTS: At the end of 4 weeks, alcohol-fed rats did not show any significant reduction in plasma or hepatic folate concentrations, plasma pyridoxal-5'-phosphate concentration, or plasma vitamin B12 concentration. On the other hand, alcohol-fed rats were significantly hyperhomocysteinemic (17.24 +/- 4.63 micromol/liter,p < 0.01) compared to the nonalcohol group (10.73 +/- 2.76 micromol/liter). Alcohol-fed rats also had a significantly lower hepatic S-adenosylmethionine and higher hepatic S-adenosylhomocysteine levels. CONCLUSIONS: Chronic alcohol consumption produces hyperhomocysteinemia by a mechanism that is related to interference with one-carbon metabolism, and not through vitamin depletion.

Analysis of folate form distribution by affinity followed by reversed- phase chromatography with electrical detection.

BACKGROUND: Naturally occurring folates exist in multiple forms, differing in pteridine ring structure and number of glutamate residues. The ability to measure these folate coenzymes in tissues and cells gives important information about in vivo folate metabolism. METHODS: Folates were heat-extracted from biological samples. A two-column HPLC system with four-channel coulometric electrochemical detection was used for analysis. An affinity column was used first to purify folates from the extract. Purified folates were eluted from the affinity column onto a phenyl analytical column, utilizing a switching valve, and folate forms were separated using an acetonitrile gradient. RESULTS: Folate forms differing in pteridine ring structure and number of glutamate chain residues were identified by retention time and characteristic response across the channels of the detector. Folates were quantified by comparison to an external calibration mixture. Limits of detection for pentaglutamyl folates ranged from 0.21 pmol for tetrahydrofolate to 0.41 pmol for 5-methyltetrahydrofolate. CVs (n = 5) for peaks containing 9-67 pmol of folate were 0.6-6.4% (within day) and 5.2-8. 4% (between days). CVs (n = 5) for peaks containing 0.9-3.5 pmol folate were 5.7-16% (within day) and 8.4-13% (between days). CONCLUSIONS: This automated HPLC system allows the simultaneous determination of polyglutamyl forms of folates from biological samples, including tetrahydrofolate, 5-methyltetrahydrofolate, formylated folates, and pteroylglutamate. The low detection limits allow analysis of folate form distribution in human samples such as erythrocytes and lymphocytes.

Effects of a physiotherapist-led stroke training programme for nurses.

AIM: to assess the effects of a physiotherapist-led stroke training programme for nurses working in a rehabilitation ward on clinical practice and patient outcome. METHOD: before and after group comparison with outcome assessment by observational and quantitative methods. Non-participant observation before and after the 5-month training programme recorded patient position, transfers and contact with nursing staff. Quantitative assessments of disability, satisfaction and mood were made at baseline, discharge and 4 months after stroke onset. We also noted selected stroke complications, rehabilitation ward length of stay and discharge destination. RESULTS: there was a significant improvement in the number of observed 'good' transfers of patients undertaken by nurses (chi2 = 9.13, df = 1, P = 0.003) but the training programme had no impact on the time the patients spent in 'poor' positions. There was no significant difference between the two groups for Barthel index scores at discharge and at 4 months. Neither was there any significant difference in the Hospital Anxiety and Depression scale, occurrence of secondary complications, length of stay or the Patient and Carer Satisfaction Questionnaires. CONCLUSION: within the limitations of the research design adopted, some improvements in clinical practice were reported but there were no significant differences in patient outcome. The training programme required no additional resources and should be replicable in most district general hospitals.

Effect of a physiotherapist-led training programme on attitudes of nurses caring for patients after stroke.

BACKGROUND: A multidisciplinary project team developed a five-month physiotherapist-led training programme designed to enhance nurses' rehabilitation skills. AIMS: Investigation of the effects of the training programme on nurses attitudes to patients with stroke. METHOD: An attitude questionnaire was completed by the participating nurses before and after the training programme. In a complementary qualitative evaluation the nurses' expectations and views of the training programme were explored. RESULTS: Analysis of the attitude questionnaire indicated that the programme was successful in positively influencing the nurses' attitudes (median of the change in score 2, p = 0.005). The qualitative interviews reflected a similar outcome. CONCLUSIONS: Quantitative and qualitative evaluation indicates that the training programme had some effect in changing nurses' attitudes to treating patients after a stroke. Such a training programme could be a useful contribution to post-stroke care.

The development of a collaborative stroke training programme for nurses.

The effectiveness of multidisciplinary stroke rehabilitation may be enhanced by nurses and therapists adopting a single consistent approach to the positioning and mobilizing of patients. As patients can spend as little as 4% of the waking day receiving 'therapy' there is considerable potential for a more dynamic nursing intervention, which may contribute to improving patient care. We aimed to investigate whether physiotherapists could step back from direct patient treatment in order to participate in a structured training programme for nurses involved with patients recovering from stroke on an established elderly care rehabilitation ward in a district general hospital. Qualitative methods were used within a participatory action research framework to describe the development process and content of the training programme. Nursing staff, physiotherapists and their respective managers were interviewed to identify perceived training needs. This informed the structure and content of the training course and allowed insight into interprofessional working.

A common mutation in the methylenetetrahydrofolate reductase gene is associated with an accumulation of formylated tetrahydrofolates in red blood cells.

A common mutation (C677T) in the gene encoding for methylenetetrahydrofolate reductase (MTHFR) (5-methyltetrahydrofolate:(acceptor) oxidoreductase, EC 1.7.99.5), a key regulatory enzyme in one-carbon metabolism, results in a thermolabile variant of the MTHFR enzyme with reduced activity in vitro. In the present study we used a chromatographic method for folate analysis to test the hypothesis that this mutation would be associated with altered distribution of red blood cell (RBC) folates. An alteration was found as manifested by the presence of formylated tetrahydrofolate polyglutamates in addition to methylated derivatives in the RBCs from homozygous mutant individuals. 5-Methyltetrahydrofolate polyglutamates were the only folate form found in RBCs from individuals with the wild-type genotype. Existence of formylated folates in RBCs only from individuals with the thermolabile MTHFR is consistent with the hypothesis that there is in vivo impairment in the activity of the thermolabile variant of MTHFR and that this impairment results in an altered distribution of RBC folates.

Limb development and evolution: a frog embryo with no apical ectodermal ridge (AER).

The treefrog Eleutherodactylus coqui is a direct developer--it has no tadpole stage. The limb buds develop earlier than in metamorphosing species (indirect developers, such as Xenopus laevis). Previous molecular studies suggest that at least some mechanisms of limb development in E. coqui are similar to those of other vertebrates and we wished to see how limb morphogenesis in this species compares with that in other vertebrates. We found that the hind limb buds are larger and more advanced than the forelimbs at all stages examined, thus differing from the typical amniote pattern. The limb buds were also small compared to those in the chick. Scanning and transmission electron microscopy showed that although the apical ectoderm is thickened, there was no apical ectodermal ridge (AER). In addition, the limb buds lacked the dorsoventral flattening seen in many amniotes. These findings could suggest a mechanical function for the AER in maintaining dorsoventral flattening, although not all data are consistent with this view. Removal of distal ectoderm from E. coqui hindlimb buds does not stop outgrowth, although it does produce anterior defects in the skeletal pattern. The defects are less severe when the excisions are performed earlier. These results contrast with the chick, in which AER excision leads to loss of distal structures. We suggest that an AER was present in the common ancestor of anurans and amniotes and has been lost in at least some direct developers including E. coqui.

Lung volume reduction surgery at a community hospital: program development and outcomes.

STUDY OBJECTIVES: Description of the development and results of a program in lung volume reduction surgery (LVRS) at a community hospital. DESIGN: Prospective data collection. SETTING: A 320-bed community hospital. PATIENTS: Fifty-five patients consecutively discharged from the hospital following LVRS. The mean preoperative FEV1 averaged 28% (+/-8%) of predicted values, while the preoperative PaCO2 averaged 49 mm Hg (+/-11.5 mm Hg). Forty-eight patients completed a preoperative conditioning regimen and underwent the procedure on an elective basis. Seven patients underwent the procedure during a hospital admission for a COPD exacerbation. Eight patients required mechanical ventilation preoperatively, including three who had required long-term mechanical ventilatory support. RESULTS: Three patients (5%) died in the hospital following surgery. One patient developed chronic ventilator dependence. All three of the patients who required long-term mechanical ventilation preoperatively were weaned from the ventilator and returned home. Follow-up pulmonary function testing is available for 42 patients 3 months after surgery, and for 20 patients 6 months after the operation. At 3 months, the mean FEV1 improved 0.19 L (p=0.0002), the mean improvement for FVC was 0.37 L (p=0.0001), and the mean drop in residual volume was 0.97 L (p=0.0001). Similar changes are seen at 6 months. Highly significant improvements were also seen in quality of life measurements and exercise performance. The benefits of surgical treatment of emphysema seemed similar in both elective and urgent groups. CONCLUSIONS: LVRS can be done safely and effectively at a community hospital, with significant improvement in pulmonary function and quality of life.

A community-based regional ventilator weaning unit: development and outcomes.

STUDY OBJECTIVE: Description of the development of a community-based weaning unit and the outcomes from that unit. DESIGN: Review of admissions, classified by etiology of ventilator dependence, with attention to disposition, length of stay, and time to wean. SETTING: Long-term acute-care facility in Worcester, Mass. PATIENTS: Two hundred seventy-eight ventilator-dependent patients admitted to a ventilator unit from 1988 through May 1995. Admissions criteria did not include prognostic considerations. INTERVENTIONS: Selected patients were entered into a formal weaning program beginning in 1992. MEASUREMENTS: Through the study period, there was a substantial growth in annual admissions, primarily due to increases in patients surviving a catastrophic acute illness. Overall, 107 of 278 (38%) patients were liberated from mechanical ventilation for at least 7 consecutive days and nights. Of the patients admitted 1993 to 1995, 31% died, 20% were discharged to a long-term care facility, 29% returned home, and 18% either remained as residents of the unit or had been transferred to acute-care facilities and were unavailable for follow-up. The highest weaning success was seen in patients with ventilator dependence from postoperative causes (58%) and acute lung injury (57%); the least success was seen in patients with ventilator dependence from COPD and neuromuscular diseases (22% each). The average time from admission to weaning fell within each diagnostic category throughout the study period. CONCLUSIONS: Rehabilitation-based ventilator weaning units play an important role in the spectrum of medical care necessary in population centers. Excellent results can result from community-based units with open admissions policies.

Evaluation and modification of an assay procedure for cysteine dioxygenase activity: high-performance liquid chromatography method for measurement of cysteine sulfinate and demonstration of physiological relevance of cysteine dioxygenase activity in cysteine catabolism.

Conflicting reports in the literature of appropriate assay procedures for measurement of cysteine dioxygenase activity led us to evaluate the procedure for assay of cysteine dioxygenase activity in rat liver preparations. Cysteine dioxygenase activity was largely in the soluble fraction of liver and was stimulated by addition of NAD+ and Fe2+. The pH optimum of the enzyme was 6.1. Addition of an inhibitor of pyridoxal 5-phosphate-dependent enzymes was necessary to prevent rapid removal of the reaction product cysteine sulfinate. Cysteine sulfinate and cysteic acid were separated by anion-exchange HPLC on a polymer-based column with trimethylamino active groups, and the reaction products were quantitated by measurement of 35S radioactivity or by formation and measurement of fluorescent derivatives. This assay of cysteine dioxygenase under optimal conditions provides a physiologically relevant measure of cysteine dioxygenase activity in liver and hepatocytes based on the observation that this activity was highly correlated with the capacity for cysteine catabolism and taurine production by isolated hepatocytes.

Rats fed a low protein diet supplemented with sulfur amino acids have increased cysteine dioxygenase activity and increased taurine production in hepatocytes.

The metabolism of cysteine and cysteinesulfinate and the activities of key enzymes in cysteine catabolic pathways were investigated in hepatocytes isolated from rats fed a basal (100 g casein/kg) diet or the diet supplemented with L-methionine (3 or 10 g/kg diet) or the sulfur equivalent as L-cystine (2.4 or 8 g/kg diet). Cysteine dioxygenase activity was higher in hepatocytes from rats fed diets with the higher level of sulfur amino acid supplementation, and the higher enzyme activity was paralleled by a greater total catabolite production (taurine + sulfate) from cysteine. Taurine production as a percentage of total cysteine catabolism was significantly greater in hepatocytes from rats fed the diet with excess methionine or cystine (basal, 22%; excess methionine, 61%, excess cystine, 49%). Glutathione production was markedly lower in hepatocytes from rats fed excess sulfur amino acids such that total cysteine utilization was similar for all dietary treatments. Cysteinesulfinate decarboxylase activity and catabolism of cysteinesulfinate by hepatocytes were unaffected by the dietary supplementations. Results are in contrast to previous studies in which increased dietary protein resulted in decreased cysteinesulfinate decarboxylase activity and decreased partitioning of cysteinesulfinate to taurine vs. sulfate. Thus, sulfur amino acids may be less effective than protein in decreasing cysteinesulfinate decarboxylase activity and may result in a pattern of sulfur catabolite production from cysteine that favors taurine production.

The activities of rat hepatic cysteine dioxygenase and cysteinesulfinate decarboxylase are regulated in a reciprocal manner in response to dietary casein level.

The catabolism of cysteine and cysteinesulfinate, the activities of key enzymes in cysteine catabolic pathways, and the effects of inhibitors of specific enzymes on cysteine catabolism were investigated in hepatocytes isolated from rats fed low (100 g casein/kg diet), moderate (300 g casein/kg diet) or high (600 g casein/kg diet) levels of dietary protein. Cysteine was catabolized predominantly by cysteinesulfinate-dependent pathways. Cysteine dioxygenase activity increased with increases in dietary casein level, and the higher enzyme activity was paralleled by a greater total catabolite production (taurine + hypotaurine + sulfate) from cysteine. However, taurine production did not closely follow cysteine dioxygenase activity. Taurine production doubled with an increase in dietary casein from 100 to 300 g/kg but did not increase with a further increase in dietary casein to 600 g/kg. Taurine production as a percentage of total catabolism decreased progressively with the increases in dietary casein and closely paralleled observed decreases in cysteinesulfinate decarboxylase activity. Thus, taurine production was limited at high protein levels by the decrease in cysteinesulfinate decarboxylase activity such that sulfate production from cysteinesulfinate was favored. D-Cysteinesulfinate inhibited cysteinesulfinate-dependent catabolism of cysteine, but inhibition of cysteinesulfinate decarboxylase was not specific.