Identification of Shell Colour Pigments in Marine Snails Clanculus pharaonius and C. margaritarius (Trochoidea; Gastropoda).

Colour and pattern are key traits with important roles in camouflage, warning and attraction. Ideally, in order to begin to understand the evolution and ecology of colour in nature, it is important to identify and, where possible, fully characterise pigments using biochemical methods. The phylum Mollusca includes some of the most beautiful exemplars of biological pigmentation, with the vivid colours of sea shells particularly prized by collectors and scientists alike. Biochemical studies of molluscan shell colour were fairly common in the last century, but few of these studies have been confirmed using modern methods and very few shell pigments have been fully characterised. Here, we use modern chemical and multi-modal spectroscopic techniques to identify two porphyrin pigments and eumelanin in the shell of marine snails Clanculus pharaonius and C margaritarius. The same porphyrins were also identified in coloured foot tissue of both species. We use high performance liquid chromatography (HPLC) to show definitively that these porphyrins are uroporphyrin I and uroporphyrin III. Evidence from confocal microscopy analyses shows that the distribution of porphyrin pigments corresponds to the striking pink-red of C. pharaonius shells, as well as pink-red dots and lines on the early whorls of C. margaritarius and yellow-brown colour of later whorls. Additional HPLC results suggest that eumelanin is likely responsible for black spots. We refer to the two differently coloured porphyrin pigments as trochopuniceus (pink-red) and trochoxouthos (yellow-brown) in order to distinguish between them. Trochopuniceus and trochoxouthos were not found in the shell of a third species of the same superfamily, Calliostoma zizyphinum, despite its superficially similar colouration, suggesting that this species has different shell pigments. These findings have important implications for the study of colour and pattern in molluscs specifically, but in other taxa more generally, since this study shows that homology of visible colour cannot be assumed without identification of pigments.

Acute intermittent porphyria and chronic renal failure.

Despite the little known association between renal damage and the acute porphyrias, limited information is available on the characteristics and pathogenesis of renal disease in this patient group. Previous reports have focused on hypertension as the principal etiological factor. We have studied a series of 9 patients with acute intermittent porphyria (AIP) attending the Porphyria Clinic at King's College Hospital, London, UK, who were referred to the Renal Unit for investigation and treatment of their renal disease. No evidence of a glomerular lesion was found in any of the patients. In contrast, renal histology showed features of a tubulointerstitial disease, and there was evidence of impaired erythropoietin production. Hypertension and nonsteroidal antiinflammatory drug use were present in about a half of the patients. It is postulated that the nephrotoxic effects of porphyrin precursors may contribute to the etiology of this clinical syndrome.

A sample distribution programme for erythropoietin.

A survey was sent to laboratories participating in the United Kingdom External Quality Assessment Service (UKNEQAS) Haematinics Scheme about the measurement of serum erythropoietin (EPO). Six laboratories, from a total of 120 that returned the survey, were measuring serum EPO concentrations by commercially available immunoassays on site in the United Kingdom. The workload of the laboratories varied from up to 100 specimens per month to more than 100 specimens analysed per week. All laboratories included control material in the assays and none of the laboratories was participating in an external quality assessment scheme for serum EPO. Four laboratories agreed to take part in the first sample distribution programme, with five and six laboratories participating in distributions 2 and 3 respectively. The results from eight kits were compared from the three distributions over a 2-year period. The serum EPO concentrations for the methods showed some variation across the range of 2.9-200 U/l when the serum EPO concentrations for each method were compared with the whole method mean. The results from this scheme have identified a role for an external quality assessment scheme for serum EPO measurements.

Erythropoietin response to hypoxia in patients with diabetic autonomic neuropathy and non-diabetic chronic renal failure.

AIMS: An erythropoietin (EPO)-deficient anaemia is recognized in Type 1 diabetic patients with early nephropathy and symptomatic autonomic neuropathy (DN). The aim of this study was to determine whether the EPO response to hypoxia was deficient in order to clarify the mechanisms involved in this process. METHODS: Five Type 1 diabetic patients DN (age 39 (28-48) years (mean (range))) with EPO-deficient anaemia (haemoglobin, Hb 10.6 (9.5-12.0) g/dl, EPO 5.0 (3.2-6.5) IU/l) and early diabetic nephropathy (persistent proteinuria 1161.6 (130-2835) mg/day, serum creatinine 97.6 (63-123) micromol/l)) were compared with nine normal subjects (age 31 (24-39) years, Hb 13.4 (11.8-15.7) g/dl, EPO 7.6 (5.6-10.3) IU/l) and four patients with non-diabetic advanced chronic renal failure RF (proteinuria 2157.5 (571-4578) mg/day, serum creatinine 490.2 (406-659) micromol/l, Hb 10.3 (9.0-11.3) g/dl, EPO 4.6 (2.9-8.5) IU/l). The subjects were exposed to 6 h of hypoxia (inspired oxygen 11.6-12.6%) by breathing a gas mixture via a hood. Hourly serum EPO levels were measured. RESULTS: All groups showed a rise in EPO production after 2 h. The diabetic DN group achieved a similar maximal response to the normal subjects at 6 h (EPO 17.3 +/-5.4 vs. 17.8 +/-7.9 IU/l). The renal failure patients mounted an EPO response to hypoxia but at lower EPO levels. CONCLUSIONS: Although the DN patients have inappropriately low EPO levels for the severity of their anaemia, they can mount an appropriate EPO response to moderate hypoxia. The mechanism underlying the EPO-deficient anaemia present in some diabetic patients remains unclear.

Anemia with erythropoietin deficiency occurs early in diabetic nephropathy.

OBJECTIVE: The normochromic normocytic anemia of erythropoietin (EPO) deficiency is recognized in advanced renal failure but not in early renal disease. The aim of this study was to determine whether anemia with EPO deficiency is found in type 1 diabetic patients with diabetic nephropathy in the absence of advanced renal failure and to compare them with patients with nondiabetic renal disease of similar severity. RESEARCH DESIGN AND METHODS: A total of 27 type 1 diabetic patients with diabetic nephropathy (DN), defined as having persistent proteinuria (mean 1,086 mg/day [CI 120-5,1901), a serum creatinine < or = 180 micromol/l, and retinopathy, were compared with 26 nondiabetic patients with glomerulonephritis (GN) and persistent proteinuria (1,874 mg/day [349-5,005]). The Hb concentration, red cell indexes, and serum EPO levels were measured, and other causes for the anemia were excluded. The EPO values were compared with a normal reference range obtained from nondiabetic patients with a microcytic anemia. The DN patients were tested for signs of diabetic peripheral and autonomic neuropathy. RESULTS: We found that 13 of the 27 DN patients were anemic (Hb 10.6 +/- 0.9 g/dl) in marked contrast to none of the GN patients (Hb 13.7 +/- 1.4 g/dl, P < 0.005). In the DN group, serum EPO concentrations failed to increase in response to anemia compared with the response seen in patients with microcytic anemia. Thus, the anemia of the DN group was associated with EPO deficiency. The anemic DN patients showed evidence of more severe proteinuria and diabetic neuropathy than the nonanemic DN patients. CONCLUSIONS: Anemia associated with EPO deficiency can occur early in DN before the onset of advanced renal failure, but does not normally occur in nondiabetic renal disease of similar severity. The pathogenesis requires elucidation.

Comparison of cardiac troponin T and I and CK-MB for the detection of minor myocardial damage during interventional cardiac procedures.

A number of cardiac interventional procedures are available for the treatment of angina, including percutaneous transluminal coronary angioplasty (PTCA), stent insertion and rotational atherectomy (RA). Variable degrees of myocardial cell injury during PTCA and stent insertion have been observed, based on rises in creatine kinase MB isoenzyme (CK-MB) and cardiac troponin T (cTnT) 6-24 h post-procedure. As there are many variations in technique within each procedure it would be helpful to be able to determine objectively the degree of myocardial damage in order to optimize technique. We measured CK-MB, cTnT and cardiac troponin I (cTnI) to ascertain which is the most sensitive marker for minor myocardial damage in this setting. Blood samples were taken both before and 6, 14 and 24h after the procedure in 109 patients (77 men) with angina, 42 of whom had unstable angina. Of the 109 patients, 86 had a stent inserted (21 as a primary stent), nine had PTCA, eight had RA and six intracoronary brachytherapy. Using the manufacturers' recommended cut-offs--CK-MB 4 microg/L, cTnI and cTnT 0.1 microg/L--five patients were excluded from further analysis as all three markers were raised pre-procedure. Post procedure all three markers were in agreement for 68 patients (44 all normal, 24 all raised). Overall, CK-MB was raised in 28 patients, cTnT in 38 and cTnI in 58. In 19 patients CK-MB and cTnT were normal, but cTnI was raised (15 between 0.11 and 0.30 microg/L). cTnI was the most sensitive indicator of minor myocardial damage, but at the recommended cut-off of 0.1 microg/L may be overly sensitive. We await the results of our follow-up study to determine the clinical implications of these small rises in cTnI.

Evaluation of six erythropoietin kits.

Six erythropoietin (EPO) measurement kits from different manufacturers were evaluated for ease of use and performance. Reference values were compared from patient samples with normal and abnormal circulating serum EPO. The kits evaluated were EPO-Trac RIA, EPORIA, DSL RIA, EPO EIA, IBL ELISA and Medac ELISA (see below for manufacturers' details). The radioimmunoassay (RIA) methods were simple to perform with little preliminary preparation of reagents but the non-isotopic methods were of varying degrees of complexity. Imprecision data showed that performance of the RIA methods was acceptable across the range 40-150 mU/mL [within-assay coefficient of variation (CV) = 2-5%, between-assay CV = 3-7%] but poor (CV > 10%) at the lower end of the range (10-15 mU/mL). The IBL enzyme-linked immunosorbent assay (ELISA) kit showed good precision across the range 10-54 mU/mL (within-assay CV = 5-7%, between-assay CV = 8-12%). The performance of the EPO enzyme immunoassay (EIA) and Medac ELISA were acceptable although precision was poor at the lower end of the range (< 10 mU/mL; within-assay CV 10% and 17% and between-assay CV 13% and 18%, respectively). The ELISA and EIA methods had lower limits of detection (0.4-0.8 mU/mL) than the RIA methods (2.3-3.6 mU/mL). Analysis of serum EPO measurement variation between methods showed evidence of significant negative bias with DSL RIA and Medac ELISA when compared with EPO-Trac RIA. Conversely, EPORIA showed significant positive bias and the two remaining methods, EPO EIA and IBL ELISA, showed no evidence of systematic bias when compared with EPO-Trac RIA. Patients with normal circulating concentration of serum EPO gave values that were within the reference range of the kits.

Comparison of the effects of theophylline and caffeine on serum erythropoietin concentration in premature infants.

UNLABELLED: Theophylline administration has been shown to attenuate erythropoietin (EP) production in adults; the effect of caffeine is not known. Our aim was to determine whether caffeine and theophylline had similar effects on EP production in the premature newborn. If caffeine was found to have a greater effect, this would influence prescribing habits. Fifty preterm infants (mean gestational age 28 weeks) who had clinically significant apnoea were randomized to receive theophylline (4 mg/kg then 2 mg/kg twice daily) or caffeine (10 mg/kg then 2.5 mg/kg once daily). The methylxanthines were continued at least until discharge from the NICU and the dosage altered to keep the levels within the therapeutic range. As an assessment of EP production, serum EP concentrations were measured. Blood for EP, haemoglobin, reticulocyte count, theophylline and caffeine levels was obtained prior to treatment and at least during weeks 3 and 7. There was no significant difference in the mean EP level in the two groups taken prior to treatment at a median age of 2 days of life. There were similar falls in haematocrit and haemoglobin in the two groups during the study period compared to pre-treatment values. At that time, however, the median reticulocyte count was higher in the caffeine compared to the theophylline treated infants (P < 0.05). This was associated with a rise compared to baseline (median 10.0-0.2 mU/ml) in the mean EP levels in the caffeine group and a decrease from a median of 10.1 to 8.3 mU/ml in the theophylline group, but the EP levels in the two groups at week 7 did not differ significantly. CONCLUSION: These results suggest that caffeine does not have a greater impact than theophylline on EP production.

The continuing need for quality assessment of cyclosporine measurement.

The returns from the United Kingdom Cyclosporin Quality Assessment Scheme were analyzed for the period June 1987 to August 1988. During this time the number of laboratories in the Scheme increased from 102 to 124 and the proportion of laboratories using nonspecific assay methods declined, as did the proportion of them measuring cyclosporine in plasma. Seven different methods were used to measure the drug in blood, and the seven methods gave seven different results when used to measure patients' samples. The results, from lowest to highest, differed by a factor of approximately 3.4. The within-assay coefficient of variation (CV) was acceptable for all methods, but the between-assay and between-center CVs were poor. HPLC gave higher CVs than did the immunoassays.

Monoclonal antibodies for radioimmunoassay of cyclosporine: a multicenter comparison of their performance with the Sandoz polyclonal radioimmunoassay kit.

The performance of a radioimmunoassay kit containing monoclonal specific and nonspecific antibodies to cyclosporine (Sandimmun-Kit; Sandoz Ltd., Basle, Switzerland) was compared with that of the original Sandoz polyclonal radioimmunoassay kit (Ciclosporin RIA-Kit). A total of 1320 blood and plasma samples from patients receiving cyclosporine after kidney, heart, liver, and bone-marrow transplantation were analyzed at six centers. For blood samples the median result on using the specific assay was about 50% of the polyclonal assay result after kidney and bone-marrow transplantation, about 33% after heart and liver transplantation; comparable figures for plasma samples were 70 and 40%. The monoclonal nonspecific-antibody assay produced results 10% to 140% higher than polyclonal-assay results, depending on sample matrix and transplant indication; the largest difference was seen in samples from heart- and liver-transplant recipients. Evidently the specific-antibody assay provides a convenient alternative to high-performance liquid chromatography for specific measurement of the drug, but the role of the new nonspecific antibody, possessing an even broader spectrum of cross-reactivity with cyclosporine metabolites than the original polyclonal antiserum, has yet to be defined.

The influence of haematocrit on blood cyclosporin measurements in vivo.

The influence of haematocrit on blood cyclosporin measurements has been studied in 276 paired blood and plasma samples from 21 renal transplant patients. A highly significant correlation was found between blood and plasma cyclosporin concentrations, r = 0.8744, but the correlation between blood or plasma cyclosporin and haematocrit was not significant. The ratio of blood/plasma cyclosporin did not significantly increase with increasing haematocrit. It was concluded that in vivo the influence of haematocrit on the measurement of blood cyclosporin concentrations was negligible.