Extracardiac 18F-florbetapir imaging in patients with systemic amyloidosis: more than hearts and minds.

PURPOSE: 18F-Florbetapir has been reported to show cardiac uptake in patients with systemic light-chain amyloidosis (AL). This study systematically assessed uptake of 18F-florbetapir in patients with proven systemic amyloidosis at sites outside the heart. METHODS: Seventeen patients with proven cardiac amyloidosis underwent 18F-florbetapir PET/CT imaging, 15 with AL and 2 with transthyretin amyloidosis (ATTR). Three patients had repeat scans. All patients had protocolized assessment at the UK National Amyloidosis Centre including imaging with 123I-serum amyloid P component (SAP). 18F-Florbetapir images were assessed for areas of increased tracer accumulation and time-uptake curves in terms of standardized uptake values (SUVmean) were produced. RESULTS: All 17 patients showed 18F-florbetapir uptake at one or more extracardiac sites. Uptake was seen in the spleen in 6 patients (35%; 6 of 9, 67%, with splenic involvement on 123I-SAP scintigraphy), in the fat in 11 (65%), in the tongue in 8 (47%), in the parotids in 8 (47%), in the masticatory muscles in 7 (41%), in the lungs in 3 (18%), and in the kidney in 2 (12%) on the late half-body images. The 18F-florbetapir spleen retention index (SRI) was calculated. SRI >0.045 had 100% sensitivity/sensitivity (in relation to 123I-SAP splenic uptake, the current standard) in detecting splenic amyloid on dynamic imaging and a sensitivity of 66.7% and a specificity of 100% on the late half-body images. Intense lung uptake was seen in three patients, one of whom had lung interstitial infiltration suggestive of amyloid deposition on previous high-resolution CT. Repeat imaging showed a stable appearance in all three patients suggesting no early impact of treatment response. CONCLUSION: 18F-Florbetapir PET/CT is a promising tool for the detection of extracardiac sites of amyloid deposition. The combination of uptake in the heart and uptake in the spleen on 18F-florbetapir PET/CT, a hallmark of AL, suggests that this tracer holds promise as a screening tool for AL.

Nephrotoxicity in survivors of Wilms' tumours in the North of England.

One aspect of concern for survivors of Wilms' tumour has been the late outcome in terms of renal function. Previous studies have documented low glomerular filtration rate and high blood pressure in some patients. Furthermore, disorders in tubular function (especially urinary concentration defects) have been suggested but not confirmed in small studies. The aim of this study was to determine the prevalence and nature of subclinical and overt glomerular, proximal and distal renal tubular toxicity in a population based cohort of survivors of Wilms' tumour. Forty patients (24 female) with a median age of 4.3 years (3 months-11.8 years) at diagnosis were studied. Median follow-up was 8.8 (range 0.06-27.5) years. Glomerular filtration rate was measured by (51)Cr-EDTA plasma clearance, proximal tubular function by electrolyte fractional excretions, urine excretion of low molecular weight proteins (retinol-binding protein) and renal tubular enzymes (alanine aminopeptidase; N-acetylglucosaminidase) and distal tubular function by the osmolality of the first two urines of the day on 3 consecutive days. Renal size (ultrasound) and blood pressure were also measured. Mean (range) glomerular filtration rate was 100 (61-150) ml min(-1) 1.73 m(-2). Nine were below the reference range for healthy individuals with two kidneys. Most serum electrolyte concentrations (sodium, potassium, chloride, calcium, magnesium and phosphate) fell within the normal range for age, as did the fractional excretions. The values that fell outside the normal range were only marginally abnormal. Subclinical measures of tubular toxicity (retinal-binding protein, alanine aminopeptidase, N-acetylglucosaminidase) were abnormal in only four patients. Thirty-seven patients achieved maximal urine osmolalities > or =800 mOsm kg(-1), but three failed to achieve this value even after DDAVP administration. Two patients had evidence of increased urinary albumin excretion. Compensatory renal hypertrophy was seen in all but two patients, but blood pressure was within normal limits in all patients. Current and past treatment for Wilms' tumour does not have any clinically important nephrotoxic effect in the majority of patients. This finding will enable paediatric oncologists to reassure patients and parents that treatment for Wilms' tumour rarely causes long-term renal impairment.

Increased sialylation of oligosaccharides on IgG paraproteins--a potential new tumour marker in multiple myeloma.

AIMS: To investigate whether changes in carbohydrate structure of IgG are related to malignancy and stage of disease in myeloma and monoclonal gammopathy of uncertain significance (MGUS). METHODS: 61 patients were studied at diagnosis: 14 with MGUS, nine with stage I multiple myeloma, 11 with stage II, 21 with stage III, and five with solitary plasmacytoma. IgG was extracted from serum by protein G affinity chromatography. Oligosaccharides were cleaved from the protein backbone enzymatically by N-glycosidase F. Oligosaccharide analysis was performed by high pressure anion exchange chromatography with pulsed electrochemical detection (HPAE-PED). RESULTS: Up to 15 oligosaccharide peaks were identified in three major fractions: neutral, monosialylated, and disialylated. Patients with myeloma showed an increase in the proportion of sialylated oligosaccharides in comparison with patients with MGUS. The ratio of neutral to sialylated oligosaccharides (N:S) was reduced at all stages of myeloma compared with MGUS: MGUS, 11.35; myeloma stage I, 7.6 (p = 0.047); stage II, 5.20 (p = 0.035); stage III, 3.60 (p = 0.0002); plasmacytoma, 7.5 (p = 0.046). The N:S ratio was independent of paraprotein concentration (r = 0.05). CONCLUSIONS: The ratio of neutral to sialylated oligosaccharides may act as a new marker of malignancy in IgG paraproteinaemia and warrants further investigation.

Glycation of monoclonal antibodies impairs their ability to bind antigen.

As elevated levels of glycated IgG have been detected in the plasma of patients with diabetes mellitus, a disease associated with increased susceptibility to infection, we have investigated whether glycation of MoAbs affects the kinetics and/or affinity of antigen binding. Three mouse MoAbs were incubated with 0.5 M glucose at pH 7.4 for 14-21 days at 37 degrees C. Control MoAbs were incubated using identical conditions but with no added glucose. Using a surface plasmon resonance technique we found that glycation significantly increased the rate of dissociation (kdiss) of the antigen-antibody complex for all three MoAbs (P < 0.05, n = 4), but had no significant effect on the rate of association (kass). For one of the MoAbs, against human IgG (Fab), we also measured kdiss by an alternative method utilizing radiolabelled antigen, which confirmed that glycation of the antibody significantly increases kdiss (P < 0.001, n = 8). We also found using an ELISA-based method that glycation of the same MoAb significantly increased the equilibrium dissociation constant (Kd) (P < 0.05, n = 6). A significant increase in kd was observed after glycation using glucose concentrations consistent with those found in poorly controlled diabetics (P < 0.02, n = 5). We conclude that in vitro glycation can significantly lower the affinity of an antibody for its antigen, and significantly increases the rate of dissociation of the antigen-antibody complex.

Serum CA 125 concentrations in patients with benign ovarian tumours.

Serum CA 125 concentrations have been measured in 115 patients with histologically confirmed nonmalignant pelvic disease, that is, serous cystadenoma (n = 56), mucinous cystadenoma (n = 14), fibroma (n = 33), thecoma (n = 8), and Brenner tumour (n = 4). Increased CA 125 concentrations (> 35 KU/L) were found in 14 patients, with a range of 46-891 KU/L, a mean of 205 KU/L, and a median of 97 KU/L. The highest values were found in patients with ascites. Serial measurements in one patient showed a fall in the 2 days immediately after surgery, over the next 3 days showing a two- to three-fold increase, followed by a slow return to normal over the next 7 weeks. Elevated CA 125 levels may not indicate ovarian malignancy and do not differentiate between benign and malignant pelvic masses.

Potential glucose sensor for perioperative blood glucose control in diabetes mellitus.

A glucose sensor intended for future use in diabetic patients undergoing major surgery is described. It involves glucose oxidase immobilised at a platinised activated carbon electrode (PACE). The sensor gave a nonoxygen dependent amperometric response in whole undiluted blood and did not require the use of electron mediators. In vitro studies in protein containing buffer using a flow cell indicated current densities of approximately 160 nA mm-2 mM-1 and a linear response over the range 0-20 mM. The operational stability of the sensor was at least 49 h in continuous use. In addition the sensor had a 90-99% response time of 1 min when used at a flow rate of 3 ml min-1 and showed a temperature dependence of 2.4% C-1. The results reported suggest significant advantages of this approach, for future use as a perioperative intra-vascular sensor for diabetic subjects, over previously reported glucose sensors.

Glycation increases the vascular clearance rate of IgG in mice.

As elevated levels of glycated IgG have been detected in the plasma of diabetics we have investigated whether glycation of IgG affects its vascular clearance rate, using a mouse model system. Polyclonal mouse IgG was aseptically incubated for 14-19 days with 0.5 M glucose in 0.1 M phosphate buffer (pH 7.4) at 37 degrees C. As control, IgG was incubated under identical conditions but with no added glucose. After incubation, both forms were labelled with 125I and injected intravenously into BALB/c mice. The rate of vascular clearance of the glycated IgG was found to be significantly higher than the control IgG in the periods 5-24 h (P < 0.001, n = 6) and 24-48 h (P < 0.01, n = 6) after injection. After 2-3 days the mice were killed and the major organs were harvested. With glycated IgG there was a significant increase in the 125I accumulated in the kidney (P < 0.02). In later experiments, dual labelling with 131I and 125I allowed mixtures of glycated and unglycated IgG to be injected into the same mouse so that the vascular clearance of both forms of IgG could be followed simultaneously. These experiments confirmed that glycation of the IgG significantly increases its vascular clearance rate.

Colorimetric assay of glycoprotein glycation free of interference from glycosylation residues.

We have developed a colorimetric assay for determining the degree of glycation of serum proteins that is unaffected by glycosylation residues. This was accomplished by reducing the proteins with sodium borohydride prior to periodate oxidation. Previous periodate-based methods, which offer several advantages over other glycation assays, cannot determine glycoprotein glycation because interference from sialic residues in the glycan chain can lead to overestimation of the amount of glycation products. Without reduction, glycation of fetuin was double that of asialofetuin glycated under identical conditions. We found that borohydride reduction before periodate oxidation increases the amount of formaldehyde released in proportion to the extent of glycation, irrespective of the degree of glycosylation. Using two glycoproteins and an unglycosylated protein, we showed how measurement of the formaldehyde increase enables the extent of glycoprotein glycation to be determined without removal of interfering sugars.

A profile of serum CA 15-3, carcinoembryonic antigen, alkaline phosphatase, and gamma-glutamyl transferase levels in patients with breast cancer.

The CA 15-3 and CEA concentrations and the alkaline phosphatase (ALP) and gamma-glutamyl transferase (Gamma GT) activities of serum from 78 patients with breast cancer have been measured. The patients included 27 with localised breast cancer, 19 who had been treated for breast cancer but were now disease-free, 17 with liver metastases, 8 with bone metastases, and 7 with disseminated breast cancer but with neither metastases to the liver or bone. As an indicator of localised breast cancer the predictive value of an increase in CA 15-3 concentration was 93%. As an indicator of metastatic breast cancer the predictive value of an increased CA 15-3 was 62%, whereas the predictive values of an increase in both CA 15-3 and CEA and an increase in CA 15-3, CEA, and ALP 88% and 100%, respectively. A normal CA 15-3 excluded metastatic breast cancer and a normal gamma-GT excluded liver metastases. The four tests together provide a set of markers for use in the follow-up of patients with breast cancer.

Phosphate efflux from jejunal enterocytes of the rat: effect of phosphate concentration gradient and pH.

The effect of variation in the intracellular and extracellular phosphate concentration on the Pi efflux across the basolateral membrane of pre-loaded enterocytes has been examined. Efflux rate constants for Pi fell from 0.89 h-1 to 0.68 h-1 as the extracellular Pi concentration was increased from 0.5 mM to 5 mM. As the intracellular Pi concentration was raised from 0.5 to 3 mM the rate constant dropped from 0.95 h-1 to 0.77 h-1. The findings are indicative of the presence of Pi-specific transporter at the basolateral membrane. The efflux rate constant of Pi at pH 7.1 was higher than that at pH 7.4 suggesting that the Pi flux across the basolateral membrane of enterocytes follows a similar pattern towards pH changes as do fluxes across the brush-border membrane.

Effect of streptozotocin-induced diabetes on the handling of phosphate by the rat small intestine.

The effect of chemically-induced diabetes on the handling of phosphate (Pi) by rat jejunal enterocytes has been investigated in the presence of a Na- or a choline-gradient. Pi uptake was significantly increased in both gradients. The Pi efflux rate constants for enterocytes from diabetic rats were similar to those of control rats. The effect of diabetes on both the protein and alkaline phosphatase isoenzymes of the rat small intestinal brush-border membranes was examined using SDS-PAGE. The patterns given by membranes from rats 14 days after the induction of diabetes were no different from those of controls.

Assay of urease-inhibiting activity in serum from children infected with Helicobacter pylori.

In order to provide a basis for obtaining further information concerning the host response to Helicobacter pylori urease, four assay methods for detecting urease-inhibiting activity in serum were examined. A quantitative assay, established in a COBAS BIO centrifugal fast analyzer and based on detection of the consumption of NADH by glutamate dehydrogenase stimulated by ammonia production, was considered most suitable for large-scale serological work. Serum samples from 63 children (aged 5 to 16 years), 28 of whom had seropositive H. pylori gastritis, were assayed. One of the serum samples in this latter group showed significant inhibitory activity. This serum sample was one of 13 in the seropositive group known to bind to urease antigen. It showed no inhibitory activity against Bacillus pasteurii or jack bean urease. Protein A binding and heat treatment indicated that the inhibitory activity was immunoglobulin G mediated. The patient from whom this sample was collected showed no distinctive features in his illness. The COBAS BIO analyzer-based urease inhibition assay provides a new tool for studying one aspect of the host response to H. pylori infection.

Assessment of chemotherapy-associated nephrotoxicity in children with cancer.

Assessment of the toxicity caused by chemotherapy in children with cancer has become more important as the number of long-term survivors has continued to increase. It is vital to monitor both acute life-threatening adverse effects and long-term toxicity that may impair the child's development and cause permanent morbidity. Renal damage may follow treatment with cytotoxic drugs, especially cisplatin or ifosfamide, and lead to glomerular, proximal tubular or distal tubular impairment or to any combination of these. Greater understanding of nephrotoxicity and of its prevention may enable the use of more intensive schedules or of higher doses of potentially nephrotoxic chemotherapy. However, the evaluation of cytotoxic drug-induced nephrotoxicity has frequently depended mainly on measurement of the plasma creatinine concentration, which may remain normal despite substantial glomerular impairment or severe tubular dysfunction. Detailed assessment of nephrotoxicity depends on an understanding of normal renal physiology and requires evaluation of all aspects of function. A comprehensive but simple investigatory protocol that enables assessment of the nature and severity of nephrotoxicity in children is described, which can be performed without admission to hospital. Glomerular function is assessed by measurement of the glomerular filtration rate from the plasma clearance of [51Cr]-ethylenediaminetetraacetic acid ([51Cr]-EDTA). Proximal nephron function is evaluated in three ways: by measurement of the concentration of calcium, magnesium, phosphate, glucose and urate in blood and urine along with calculations of their fractional excretion and of the renal threshold for phosphate; by determination of the excretion in urine of low-molecular-weight proteins (e.g. retinol-binding protein); and by investigation of urinary bicarbonate excretion in patients who are acidotic. Distal nephron function is initially investigated by examination of the concentration (osmolality) and acidification (pH) of an early morning sample of urine. Finally, a group of general investigations is performed, including quantitation of urinary excretion of renal tubular enzymes (e.g. N-acetylglucosaminidase) and measurement of blood pressure.

Serum CA 12-5 concentrations and CA 12-5/CEA ratios in patients with epithelial ovarian cancer.

In attempts to increase the specificity of the CA 12-5 test the ratio of CA 12-5 and CEA concentrations has been determined in 155 cancer patients, all of whom had an increased serum CA 12-5. The patients included 47 with epithelial ovarian cancer, 38 with colorectal cancer, 24 with cervical cancer, 20 with lung cancer, 17 with gastric cancer, and 9 with pancreatic cancer. The CA 12-5/CEA ratio in serum of patients with ovarian cancer ranged from 30 to 920 (mean 251), whereas in other types of cancer the highest ratio was 240 and the mean was 13. All 47 patients with ovarian cancer, but only 7 of the 108 patients with other types of cancer, showed a CA 12-5/CEA ratio greater than 25. About 10% of the patients with gastric or colorectal cancer but none of those with other types of cancer showed an increased ratio. As the predictive value of a CA 12-5/CEA ratio of less than 25 excluding ovarian cancer is 100%, we recommend measuring the CEA concentration in all those with increased CA 12-5 and calculation of the CA 12-5/CEA ratio.

Neuron-specific enolase during chemotherapy of small cell lung cancer.

Serum neuron-specific enolase (NSE) has been measured in 28 patients with small cell lung cancer (SCLC) and 90 patients with other forms of lung cancer (NSCLC), i.e., 28 with adenocarcinoma and 62 with squamous cell carcinoma. Increased NSE (greater than 12.0 micrograms/liter) was found in 71.4% of SCLC patients and in 22.2% of NSCLC patients. The predictive value of an increased NSE in identifying SCLC was only 50%, whereas the predictive value of a normal NSE in differentiating SCLC for NSCLC was 91%. Serial studies during chemotherapy of SCLC patients showed that the doubling time of NSE ranged from 7 to 127 days and the mean apparent half-life (AHL) of NSE to be 14 days. AHL values in excess of 20 days suggest that the tumour is not in full remission. We believe that measurement of serum NSE and calculation of the AHL and DT are valuable in identifying the effectiveness of chemotherapy in patients with SCLC.