Cardiorespiratory fitness is impaired and predicts mid-term postoperative survival in patients with abdominal aortic aneurysm disease.

NEW FINDINGS: What is the central question of this study? To what extent cardiorespiratory fitness is impaired in patients with abdominal aortic aneurysmal (AAA) disease and corresponding implications for postoperative survival requires further investigation. What is the main finding and its importance? Cardiorespiratory fitness is impaired in patients with AAA disease. Patients with peak oxygen uptake of <13.1 ml O2  kg-1  min-1 and ventilatory equivalent for carbon dioxide at anaerobic threshold ≥34 are associated with increased risk of postoperative mortality at 2 years. These findings demonstrate that cardiorespiratory fitness can predict mid-term postoperative survival in AAA patients, which may help to direct care provision. ABSTRACT: Preoperative cardiopulmonary exercise testing is a standard assessment of cardiorespiratory fitness (CRF) and risk stratification. However, to what extent CRF is impaired in patients undergoing surgical repair of abdominal aortic aneurysm (AAA) disease and the corresponding implications for postoperative outcome requires further investigation. We measured CRF during an incremental exercise test to exhaustion using online respiratory gas analysis in patients with AAA disease (n = 124, aged 72 ± 7 years) and healthy sedentary control subjects (n = 104, aged 70 ± 7 years). Postoperative survival was examined for association with CRF, and threshold values were calculated for independent predictors of mortality. Patients who underwent preoperative cardiopulmonary exercise testing before surgical repair had lower CRF [age-adjusted mean difference of 12.5 ml O2  kg-1  min-1 for peak oxygen uptake ( V ̇ O 2 peak ), P < 0.001 versus control subjects]. After multivariable analysis, both V ̇ O 2 peak and the ventilatory equivalent for carbon dioxide at anaerobic threshold ( V ̇ E / V ̇ C O 2 - AT ) were independent predictors of mid-term postoperative survival (2 years). Hazard ratios of 5.27 (95% confidence interval 1.62-17.14, P = 0.006) and 3.26 (95% confidence interval 1.00-10.59, P = 0.049) were observed for V ̇ O 2 peak  < 13.1 ml O2  kg-1  min-1 and V ̇ E / V ̇ C O 2 - AT ≥ 34, respectively. Thus, CRF is lower in patients with AAA, and those with a V ̇ O 2 peak  < 13.1 ml O2  kg-1  min-1 and V ̇ E / V ̇ C O 2 - AT ≥ 34 are associated with a markedly increased risk of postoperative mortality. Collectively, our findings demonstrate that CRF can predict mid-term postoperative survival in AAA patients, which may help to direct care provision.

The cardiopulmonary exercise test grey zone; optimising fitness stratification by application of critical difference.

BACKGROUND: Cardiorespiratory fitness can inform patient care, although to what extent natural variation in CRF influences clinical practice remains to be established. We calculated natural variation for cardiopulmonary exercise test (CPET) metrics, which may have implications for fitness stratification. METHODS: In a two-armed experiment, critical difference comprising analytical imprecision and biological variation was calculated for cardiorespiratory fitness and thus defined the magnitude of change required to claim a clinically meaningful change. This metric was retrospectively applied to 213 patients scheduled for colorectal surgery. These patients underwent CPET and the potential for misclassification of fitness was calculated. We created a model with boundaries inclusive of natural variation [critical difference applied to oxygen uptake at anaerobic threshold (V˙O2-AT): 11 ml O2 kg-1 min-1, peak oxygen uptake (V˙O2 peak): 16 ml O2 kg-1 min-1, and ventilatory equivalent for carbon dioxide at AT (V̇E/V̇CO2-AT): 36]. RESULTS: The critical difference for V˙O2-AT, V˙O2 peak, and V˙E/V˙CO2-AT was 19%, 13%, and 10%, respectively, resulting in false negative and false positive rates of up to 28% and 32% for unfit patients. Our model identified boundaries for unfit and fit patients: AT <9.2 and ≥13.6 ml O2 kg-1 min-1, V˙O2 peak <14.2 and ≥18.3 ml kg-1 min-1, V˙E/V˙CO2-AT ≥40.1 and <32.7, between which an area of indeterminate-fitness was established. With natural variation considered, up to 60% of patients presented with indeterminate-fitness. CONCLUSIONS: These findings support a reappraisal of current clinical interpretation of cardiorespiratory fitness highlighting the potential for incorrect fitness stratification when natural variation is not accounted for.

Examination of fine-scale spatial-temporal overlap and segregation between two closely related congeners Gadus morhua and Gadus ogac in coastal Newfoundland.

The spatial and temporal movement patterns of sympatric juvenile Atlantic cod Gadus morhua and Greenland cod Gadus ogac were studied using high-resolution radio-acoustic positioning in a coastal area of Newfoundland during the summers of 2009 and 2010. A total of 20 fish (10 G. ogac and 10 G. morhua) were equipped with acoustic transmitters and monitored for periods up to 23 days. Most fishes showed high site fidelity with mean residence times of 12·4 (G. morhua) and 14·4 days (G. ogac). A few individuals showed a transient use of the study area, ranging distances up to c. 4 km. Mean daily home ranges [95% kernel utilization distributions (KUDs)] and core activity areas were significantly larger for G. morhua (3·8 and 0·5 ha) than for G. ogac (2·7 and 0·3 ha). Home ranges were not related to total length (LT ) for G. morhua but showed a weak positive relationship for G. ogac. Gadus morhua occupied larger areas during the day while G. ogac occupied slightly larger areas at night. Mean rates of movement for G. ogac and G. morhua ranged from 0·83 to 1·24 and 0·76 to 1·76 LT s(-1) , respectively, and were highest during crepuscular periods. Overall, G. morhua were wider ranging, moved at faster rates and were active throughout the diel cycle compared to G. ogac of the same size. It is suggested that differential use of space and activity periods plays an important role in the successful coexistence of these two species.

Short, natural, and extended photoperiod response in BC2F4 lines of bread wheat with different photoperiod-1 (Ppd-1) alleles.

Flowering is a critical period in the life cycle of flowering plant species, resulting in an irreversible commitment of significant resources. Wheat is photoperiod sensitive, flowering only when daylength surpasses a critical length; however, photoperiod insensitivity (PI) has been selected by plant breeders for >40 years to enhance yield in certain environments. Control of flowering time has been greatly facilitated by the development of molecular markers for the Photoperiod-1 (Ppd-1) homeoloci, on the group 2 chromosomes. In the current study, an allelic series of BC2F4 lines in the winter wheat cultivars 'Robigus' and 'Alchemy' was developed to elucidate the influence on flowering of eight gene variants from the B- and D-genomes of bread wheat and the A-genome of durum wheat. Allele effects were tested in short, natural, and extended photoperiods in the field and controlled environments. Across genetic background and treatment, the D-genome PI allele, Ppd-D1a, had a more potent effect on reducing flowering time than Ppd-B1a. However, there was significant donor allele effect for both Ppd-D1a and Ppd-B1a, suggesting the presence of linked modifier genes and/or additional sources of latent sensitivity. Development of Ppd-A1a BC2F4 lines derived from synthetic hexaploid wheat provided an opportunity to compare directly the flowering time effect of the A-genome allele from durum with the B- and D-genome variants from bread wheat for the first time. Analyses indicated that the reducing effect of Ppd-A1a is comparable with that of Ppd-D1a, confirming it as a useful alternative source of PI.

Strategy for exploiting exotic germplasm using genetic, morphological, and environmental diversity: the Aegilops tauschii Coss. example.

Hexaploid bread wheat evolved from a rare hybridisation, which resulted in a loss of genetic diversity in the wheat D-genome with respect to the ancestral donor, Aegilops tauschii. Novel genetic variation can be introduced into modern wheat by recreating the above hybridisation; however, the information associated with the Ae. tauschii accessions in germplasm collections is limited, making rational selection of accessions into a re-synthesis programme difficult. We describe methodologies to identify novel diversity from Ae. tauschii accessions that combines Bayesian analysis of genotypic data, sub-species diversity and geographic information that summarises variation in climate and habitat at the collection point for each accession. Comparisons were made between diversity discovered amongst a panel of Ae. tauschii accessions, bread wheat varieties and lines from the CIMMYT synthetic hexaploid wheat programme. The selection of Ae. tauschii accessions based on differing approaches had significant effect on diversity within each set. Our results suggest that a strategy that combines several criteria will be most effective in maximising the sampled variation across multiple parameters. The analysis of multiple layers of variation in ex situ Ae. tauschii collections allows for an informed and rational approach to the inclusion of wild relatives into crop breeding programmes.

Cardiorespiratory fitness fails to predict short-term postoperative mortality in patients undergoing elective open surgery for abdominal aortic aneurysm.

INTRODUCTION: Preoperative cardiopulmonary exercise testing aids surgical risk stratification and is an established predictor of mid- to long-term survival in patients undergoing elective open abdominal aortic aneurysm repair. Whether cardiopulmonary exercise testing also predicts 30-day mortality in this population remains to be established. MATERIALS AND METHODS: Data for 109 patients (mean age 72 years) who underwent cardiopulmonary exercise testing to assess risk for surgical abdominal aortic aneurysm repair was analysed. Patients were classified according to cardiopulmonary fitness as fit (peak oxygen uptake ≥ 15ml O2.kg-1.min-1) or unfit (peak oxygen uptake less than 15ml O2.kg-1.min-1) and further stratified according to clamp position (infrarenal or suprarenal). Between-group postoperative outcomes were compared for in-hospital 30-day mortality, postoperative morbidity scale scores (day 5) and hospital length of stay. RESULTS: Seventy-nine patients underwent open surgery and 30 patients were treated conservatively. No deaths were recorded at 30 days post-surgery. Unfit patients with infrarenal clamping exhibited higher postoperative morbidity scale scores (64% vs 26%) and longer length of stay (four days) than fit patients (p < 0.05). Conversely, with suprarenal clamping, postoperative morbidity scale scores were similar and length of stay longer (three days) in fit compared with unfit patients (p < 0.05). DISCUSSION AND CONCLUSION: Preoperative fitness level defined by peak oxygen uptake failed to identify patients at risk of 30-day mortality when undergoing elective abdominal aortic aneurysm repair. Postoperative morbidity and length of stay in patients with suprarenal clamping was high independent of cardiopulmonary fitness. These findings suggest that cardiopulmonary exercise testing may be a useful predictor of complications following infrarenal rather than suprarenal clamping but may not be a good predictor of 30-day mortality.

Effects of prolonging peak dobutamine dose during stress echocardiography.

OBJECTIVES: This study sought to test whether the physiologic advantage of a prolonged dobutamine stage during stress echocardiography can be effectively combined with a clinically practical infusion protocol. BACKGROUND: Dobutamine has a half-life of 2 min and requires up to 10 min to achieve steady state. Despite these known pharmacodynamics, dobutamine stress echocardiography is routinely performed by advancing doses at 3-min intervals. Canine studies have shown that dobutamine stress echocardiography end points will occur at a lower dose if each stage is prolonged, but these findings have yet to be used in the clinical setting. METHODS: The standard 3-min dobutamine dose stage during stress echocardiography was modified by extending the peak dose (40 micrograms/kg body weight per min) for an additional 2 min. Consecutive patients underwent this modified protocol to test whether the requirement for atropine could be reduced. According to this modified protocol, if a dobutamine stress echocardiographic end point (85% of maximal predicted heart rate, new wall motion abnormalities, hypotension, arrhythmia or intolerable symptoms) was not reached at 3 min of the peak dose, this dose was prolonged for an additional 2 min. If a doubtamine stress echocardiographic end point was still not attained, atropine (up to 1.0 mg intravenously) was administered. RESULTS: The study included 84 patients, 22 of whom (26.2%) achieved a dobutamine stress echocardiographic end point using the standard 3-min stage. Of the 62 patients who did not reach an end point in the initial 3 min of peak dobutamine dose, the additional 2 min of dobutamine increased heart rate (from 99.6 +/- 23.8 to 107.2 +/- 23.2 beats/min, p < 0.01) and allowed 20 patients (32.3%, p < 0.01) to attain an end point. Of the remaining 42 patients, 23 never achieved a stress echocardiographic end point, despite 1.0 mg of atropine. One patient developed supraventricular tachycardia during the additional 2 min of dobutamine, and one developed nonsustained ventricular tachycardia after receiving atropine. CONCLUSIONS: These data demonstrate that a significant number of patients (32%) who do not reach a dobutamine stress echocardiographic end point with the standard protocol can safely attain an end point solely by extending the duration of the peak dose. Adoption of this strategy may reduce the need for supplemental atropine and its potential adverse effects.

Coronary endothelial dysfunction in patients with acute-onset idiopathic dilated cardiomyopathy.

OBJECTIVES: This study sought to determine whether coronary endothelial dysfunction exists in patients with acute-onset idiopathic dilated cardiomyopathy (DCM) and to explore its relation to recovery of left ventricular systolic function in this patient population. BACKGROUND: Coronary endothelial dysfunction exists in chronic DCM, but its importance in the development and progression of ventricular dysfunction is not known. To address this issue we studied coronary endothelial function in patients with idiopathic DCM <6 months in duration and explored the relation between coronary endothelial function and subsequent changes in left ventricular ejection fraction (LVEF). METHODS: Ten patients with acute-onset idiopathic DCM (duration of heart failure symptoms 2.0 +/- 0.4 months [mean +/- SEM]) and 11 control patients with normal left ventricular function underwent assessment of coronary endothelial function during intracoronary administration of the endothelium-dependent vasodilator acetylcholine and the endothelium-independent vasodilator adenosine. Coronary cross-sectional area (CSA) was determined by quantitative coronary angiography and coronary blood flow (CBF) by the product of coronary CSA and CBF velocity measured by an intracoronary Doppler catheter. Patients with DCM underwent assessment of left ventricular function before and several months after the study. RESULTS: Acetylcholine infusion produced no change in coronary CSA in control patients but significant epicardial constriction in patients with DCM (-36 +/- 11%, p < 0.01). These changes were associated with increases in CBF in control patients (+118 +/- 49%, p < 0.01) but no change in patients with DCM. Infusion of adenosine produced increases in coronary caliber and blood flow in both groups. Follow-up assessment of left ventricular function was obtained in nine patients with DCM 7.0 +/- 1.7 months after initial study, at which time LVEF had improved by > or =0.10 in four patients. Multiple linear regression revealed a positive correlation between both the coronary CSA (r2 = 0.57, p < 0.05) and CBF (r2 = 0.68, p < 0.01) response to acetylcholine and the subsequent improvement in LVEF. CONCLUSIONS: Coronary endothelial dysfunction exists at both the microvascular and the epicardial level in patients with acute-onset idiopathic DCM. The preservation of coronary endothelial function in this population is associated with subsequent improvement in left ventricular function.

Accurate localization of mitral regurgitant defects using multiplane transesophageal echocardiography.

BACKGROUND: Appropriate patient selection for surgical repair of the mitral valve depends on the specific location and mechanism of regurgitation, which, in turn, has necessitated a more detailed method to accurately describe mitral pathology. This study tests a strategy of using multiplane transesophageal echocardiography to systematically localize mitral regurgitant defects and compares these results with the surgical findings. METHODS: Fifty patients with mitral regurgitation underwent intraoperative transesophageal echocardiography for the evaluation of mitral pathology and potential repair. Mitral regurgitant defects were localized using a systematic strategy and a simple nomenclature that divides each mitral valve into six sections (three sections per leaflet) and each prosthetic sewing ring into six sections (60 radial degrees = one section). RESULTS: Thirty-nine patients with native mitral valves were studied, for a total of 234 sections evaluated. Eighty-seven of these sections contained regurgitant defects by transesophageal echocardiography (mean number of regurgitant defects per valve, 2.2; range, 1 through 6). There was agreement between the transesophageal echocardiographic and surgical localizations in 96% (224/234; p < 0.0001) of the sections. Eleven patients with prosthetic mitral valves were studied, for a total of 66 sections evaluated. Twenty-three of these sections contained paravalvular leaks by transesophageal echocardiography (mean number of leaks per prosthesis, 2.1; range, 1 through 6). There was agreement between the transesophageal echocardiographic and surgical localizations in 88% (58/66; p < 0.001) of the sections. CONCLUSIONS: This transesophageal echocardiographic strategy provides a systematic method to accurately localize mitral regurgitant lesions and has the potential to improve the preoperative assessment of patients with significant mitral regurgitation.

Abnormal global left ventricular relaxation occurs early during the development of pharmacologically induced ischemia.

In animal and human models, left ventricular (LV) diastolic function has been observed to be highly sensitive to myocardial ischemia. The response of LV diastolic parameters to pharmacologically induced ischemia, however, has not been characterized and might be important in the interpretation of dobutamine stress echocardiography. Eight mongrel dogs, in which were inserted a high-fidelity micromanometer LV catheter, coronary sinus sampling catheter, and ultrasonic coronary artery flow probe, underwent intravenous dobutamine infusion at escalating doses both before (control protocol) and after (ischemia protocol) creation of left anterior descending coronary artery stenosis with a hydraulic cuff occluder adjusted to maintain resting coronary artery flow but attenuate reactive hyperemia. At each dobutamine dose, epicardial short-axis 2-dimensional echocardiographic images and hemodynamic measurements were obtained. LV diastolic function was examined by calculation of peak (-)dP/dt and the time constant of isovolumic relaxation (tau). The dobutamine infusion protocol was terminated on the earliest recognition of an anterior wall motion abnormality. Peak (+)dP/dt normalized for developed isovolumetric pressure was calculated as a relatively load-independent index of global LV contractile function. Dobutamine infusion with and without ischemia resulted in comparable changes in heart rate and (+)dP/dt/IP, with no change in LV end-diastolic or -systolic pressure. The magnitude of peak (-)dP/dt increased less during the ischemia (1231 +/- 109 to 1791 +/- 200 mm Hg/sec) versus the control (1390 +/- 154 to 2432 +/- 320 mm Hg/sec) protocol (P <.05). Similarly, the observed decrease in tau was less during the ischemia (53 +/- 3 to 38 +/- 4 msec) than the control (51 +/- 5 to 23 +/- 3 msec) protocol, corresponding to a slower rate of relaxation (P <.05). In addition, the smaller decrease in tau was observed at the dobutamine dose before the dose at which an echocardiographic wall motion abnormality was first recognized. Dobutamine-induced ischemia is associated with abnormal LV diastolic function. In addition, these abnormalities seem to occur early in the development of ischemia. These observations extend to pharmacologically induced ischemia prior findings from other models of ischemia, suggesting the high sensitivity of LV diastolic function to the development of myocardial ischemia.

Medical investigation and treatment of urinary stones: a search for new ideas.

The main threads of recent research into the cause and treatment of calcium oxalate/phosphate stones are reviewed. Little progress has been made in recent years, and two main reasons can be identified. First, there has been too much emphasis upon calcium and too little emphasis upon urinary oxalate as causative factors. Second, there has been too much emphasis upon the physical chemistry of simple solutions and too little emphasis upon the study of whole urine.

A new enzymatic method for the determination of glycollate in urine and plasma.

An enzymatic assay for the measurement of glycollate in urine and plasma is described. Gycollic acid oxidase (glycollate:oxygen oxidoreductase, EC 1.1.3), extracted from spinach leaves, is used in the enzymatic oxidation of glycollate to produce glyoxylate and hydrogen peroxide. In the presence of peroxidase, the hydrogen peroxide oxidatively couples sulphonated 2,4-dichlorophenol and 4-aminophenazone to form a soluble purple quinoneimine dye. Interfering substances are removed from plasma by deproteinisation and from urine by adsorption onto activated charcoal before analysis. Glycollic acid oxidase also catalyses the oxidation of lactic acid which therefore has to be determined separately. The mean normal urinary glycollate for adults is 0.19 mmol/24 h with no difference between males and females. The mean normal plasma glycollate for adults is 0.17 mmol/l, but higher in males than in females.

The real and apparent plasma oxalate.

A method for the determination of human plasma oxalate concentration by an enzymatic assay procedure is described using deproteinised plasma. The apparent concentration of oxalate in 20 normal subjects was 1.1--16.0 mumol/l (mean 7.78; S.D. 3.96). It was suspected that these results might be too high, due to the possible conversion of glyoxalate to oxalate, and this reaction was clearly demonstrated to occur in whole blood in vitro. Inhibitors (boric acid, DL-beta-phenyllactic acid, and allopurinol) of this oxidation were therefore added to the freshly taken blood samples, prior to assaying by the same technique. The plasma oxalate concentration from normal subjects was then found to be 0--5.4 mumol/l (mean 2.26; S.D. 1.67). It is concluded that normal blood spontaneously generates oxalate on standing, and the higher values obtained by other in vitro methods must be fallacious.

Measurement of plasma oxalate in healthy subjects and in patients with chronic renal failure using immobilised oxalate oxidase.

A continuous flow assay using immobilised oxalate oxidase was used to measure the level of oxalate in plasma ultrafiltrate obtained from healthy subjects and from patients with chronic renal failure. The levels of oxalate in plasma from normal subjects ranged from 1.3-3.1 mumol/l (mean 2.03; SD = 0.52) with females showing a higher (p less than 0.05) level (mean 2.25 mumol/l) than males (mean 1.87 mumol/l). The mean oxalate/creatinine clearance ratio in fourteen healthy subjects was greater than unity, thus indicating a net tubular secretion of oxalate. At physiological pH, L-ascorbate was converted to oxalate in whole blood following venepuncture, in plasma and in plasma ultrafiltrate. Reduction of the spontaneous generation of oxalate in the samples prior to analysis was achieved by acidification and treatment with sodium nitrite. A linear correlation (r = 0.92; p less than 0.001) was found between plasma oxalate and plasma creatinine in patients with chronic renal failure.

Liquid chromatographic studies of vitamin B6 metabolism in man.

The effects of increased intake of pyridoxine hydrochloride on plasma vitamin B6 metabolism within therapeutic limits (up to 800 mg/day) were investigated. Maximum plasma concentrations of pyridoxal phosphate were attained at relatively low intakes of pyridoxine hydrochloride. Two metabolism thought to be unidentified forms of vitamin B6 were present in subjects taking more than 200 mg of pyridoxine hydrochloride per day as have recently been described. We investigated the possibility that these were isomeric forms of vitamin B6. However, 'Peak 2' metabolite was shown to be probably 4-pyridoxolactone. The metabolism of isopyridoxal has not previously been investigated in man. We demonstrated that it is an active vitamer of the B6 complex in humans. The main fluorescent metabolite of isopyridoxal present in plasma and urine had a similar retention time to 'Peak 1' metabolite. Isopyridoxal was incapable of being directly phosphorylated in rat liver extract and it is therefore unlikely that peak 1 is isopyridoxal phosphate. Its nature remains unknown.

Plasma oxalate and creatinine and oxalate/creatinine clearance ratios in normal subjects and in primary hyperoxaluria. Evidence for renal hyperoxaluria.

Plasma oxalate and creatinine were measured repeatedly in healthy individuals and in 12 patients with type 1 primary hyperoxaluria unresponsive to pyridoxine. The mean ratios were 0.025 (SD 0.006) and 0.120 (SD 0.048), respectively. One patient repeatedly had normal plasma oxalate despite markedly raised urinary oxalate and it seems unlikely that this excess oxalate could have come from the liver. Oxalate/creatinine clearance ratios in the normal group had an overall mean of 0.59 (SD 0.27) in 24 h urine collections and 0.741 (SD 0.297) in repeated short clearance periods. Both renal tubular absorption and secretion of oxalate apparently occurred on different days, but this did not depend upon urinary flow rate. Oxalate/creatinine clearance ratios in type 1 primary hyperoxaluria had a mean of 2.88 (SD 3.11). The raised oxalate/creatinine clearance ratios in the patients were not correlated with either plasma oxalate or creatinine. A few patients showed much higher clearance ratios and in some were sufficiently high to indicate that oxalate was generated and secreted in the kidneys.

Continuous-flow assay for urinary oxalate using immobilised oxalate oxidase.

A continuous-flow assay for measuring oxalate in urine is described. Covalently attached oxalate oxidase (EC 1.2.3.4) is used to oxidize the oxalate anion to carbon dioxide and hydrogen peroxide. The formed hydrogen peroxide is measured colorimetrically (A580) with an established reaction using horseradish peroxidase (EC 1.11.17), 3-methyl-2-benzothiazolinone hydrazone (MBTH) and 3-dimethylaminobenzoic acid (DMAB). Ascorbate interference is eliminated by treating the urine sample with sodium nitrite prior to assaying. The assay is accurate (mean recovery of added oxalate in spiked urine sample is 93 +/- 11%), sensitive (detection limit 1.0 mumol/L), reproducible (within-batch CV 3.5%; between-batch CV 5%) and relatively rapid (15 samples/h). This assay correlates well (R = 0.99) with another established enzymatic method (using oxalate decarboxylase).

Determination of plasma pyridoxal phosphate levels using a modified apotryptophanase assay.

The development of a simple non-isotopic enzymatic assay for plasma pyridoxal phosphate (PLP) using a modified apotryptophanase procedure is reported. Recovery of added PLP from plasma was 86% and the within-run precision of the assay was 6.5%. Between-run precision was 7.5%. Concentrations of cupric ions as low as 20 mumol/L severely inhibited apotryptophanase activity and this effect of copper was almost completely prevented by the addition of EDTA. The reference plasma PLP range obtained by the apotryptophanase method was found to be between 5 and 50 micrograms/L plasma, with a mean of 17 +/- 10 micrograms/L plasma. The assay requires minimum technical skill and, with the aid of a multisample reaction rate analyser, more than 50 samples could easily be handled within a working day.