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.

Optimized methods to measure acetoacetate, 3-hydroxybutyrate, glycerol, alanine, pyruvate, lactate and glucose in human blood using a centrifugal analyser with a fluorimetric attachment.

Optimized methods are described for the analysis of glucose, lactate, pyruvate, alanine, glycerol, D-3-hydroxybutyrate and acetoacetate in perchloric acid extracts of human blood using the Cobas Bio centrifugal analyser. Glucose and lactate are measured using the photometric mode and other metabolites using the fluorimetric mode. The intra-assay coefficients of variation ranged from 0.7 to 4.1%, except with very low levels of pyruvate and acetoacetate where the coefficients of variation were 7.1 and 12% respectively. All seven metabolites can be measured in a perchloric acid extract of 20 mul of blood. The methods have been optimized with regard to variation in the perchloric acid content of the samples. These variations arise from the method of sample preparation used to minimize changes occurring in metabolite concentration after venepuncture.

Assessment of renal toxicity by urinary enzymes in patients receiving chemotherapy with 8-methyl-8-acetylenic-putrescine.

Renal toxicity was assessed in 19 patients receiving methyl acetylenic putrescine (MAP), an irreversible inhibitor of ornithine decarboxylase. Patients received 250 mg t.d.s. for up to 13 weeks. This dose effectively inhibited the target enzyme, as shown by elevations in decarboxylated S-adenosyl methionine levels. No significant nephrotoxicity was observed in these patients as determined by plasma urea, creatinine and creatinine clearance measurements, although minor elevations of the urinary enzymes lactate dehydrogenase, N-acetyl-beta-glucosaminidase, alkaline phosphatase and alanine aminopeptidase were observed. As this could represent sub-clinical renal damage, caution should be exercised when using MAP in combination with other cytotoxic drugs.

Blood glucose, lactate, pyruvate, glycerol, 3-hydroxybutyrate and acetoacetate measurements in man using a centrifugal analyser with a fluorimetric attachment.

Methods are described for the analysis of glucose, lactate, pyruvate, alanine, glycerol, 3-hydroxybutyrate and acetoacetate in perchloric acid extracts of human blood, using the Cobas Bio centrifugal analyser fitted with a fluorimetric attachment. Intra-assay and inter-assay coefficients of variation ranged from 1.9 to 7.9% and from 1.0 to 7.2% respectively. Correlation coefficients ranged from 0.96 to 0.99 against established continuous-flow and manual spectrophotometric methods. All seven metabolites can be measured using a single perchloric acid extract of 20 microliter of blood. The versatility of the assays is such that as little as 100 pmol pyruvate, 3-hydroxybutyrate or as much as 15 nmol glucose can be measured in the same 20 microliter extract.

Comparison of diazo-coupling, formazan, and silver staining techniques for visualizing alkaline phosphatase isoenzymes after electrophoresis in homogeneous-pore and gradient-pore polyacrylamide gels.

Three techniques for visualization of alkaline phosphatase after polyacrylamide-gel electrophoresis are compared. These are diazo-dye simultaneous coupling with the substrate sodium naphthyl phosphate and 5-chloro-2-toluene diazonium chloride; formazan precipitation with the substrate 5-bromo-4-chloro-3-indolyl phosphate and 3-[4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; and silver staining with the substrate sodium glycerophosphate. Each staining technique was tested with gradient-pore and homogeneous-pore acrylamide-gel electrophoresis. The main factors assessed are sensitivity; separation of the human serum alkaline phosphatase isoenzymes of the liver, bone, and intestinal types; and differences in substrate affinity, as well as the complexity of each technique. Using the three techniques only minor differences in substrate affinity are evident. There is some nonspecific staining with the diazo-coupling technique but not with the formazan and silver staining techniques. The differences, in the mobility of the liver, bone, and intestinal isoenzymes achieved by homogeneous-pore gel electrophoresis are sufficient to allow them to be clearly distinguished. However, only very small differences in mobility are found with gradient-pore gel electrophoresis, but the sharper bands in this medium allow much smaller amounts of activity to be detected. As little as 160 microU of enzyme can be visualized by the diazo technique. Silver staining gives an approximately fourfold increase in sensitivity over the formazan technique, which in turn gives a fourfold increase over the diazo technique. An important aspect of the silver staining technique is the potential of increasing sensitivity much further by improvements in the photographic physical development stage.(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary protein and enzyme excretion in patients receiving chemotherapy with the cis-platinum analogs carboplatin (CBDCA, JM8) and iproplatin (CHIP, JM9).

Urinary protein and enzyme excretion was measured in 33 patients with solid tumours receiving chemotherapy with the cis-platinum analogs carboplatin (JM8, CBDCA) and iproplatin (JM9, CHIP). The patients were given up to six courses of the drugs at 4-week intervals, and serial urine samples were collected weekly for periods up to 28 weeks. Overall there was no significant increase in the alkaline phosphatase (ALP), lactate dehydrogenase (LD), and N-acetyl glucosaminidase (NAG) excretion of the first posttreatment samples compared with the pretreatment samples. During the course of treatment there were transient increases in all three enzymes, some quite marked. There was no consistent increase in urinary protein or enzyme excretion during the period of treatment, suggesting that there was no cumulative nephrotoxicity. There was no change in creatinine clearance or urinary beta 2-microglobulin content. Iproplatin appeared marginally more toxic on the basis of elevated NAG and ALP during the second half of the treatment periods compared with the first (P less than 0.01 and less than 0.025, respectively).

Urinary enzyme excretion after donor nephrectomy.

A number of recent studies of long-term kidney donors have reviewed glomerular function and blood pressure. Little attention has been paid to the potentially damaging effects of compensatory hyperfiltration on renal tubular cells after donor nephrectomy. The urinary excretion of high-molecular-weight enzymes is a sensitive indicator of renal tubular cell damage. This study compares the urinary excretion of four enzymes (alanine aminopeptidase, alkaline phosphatase, N-acetyl-beta-D-glucosaminidase, and lactate dehydrogenase) in a group of 77 subjects who had undergone unilateral nephrectomy up to 21 years previously with 52 healthy non-nephrectomized controls. The urinary excretion for all four enzymes by the remaining kidney after contralateral nephrectomy in the kidney donors was significantly greater than the enzyme excretion per single kidney in the control group (p less than 0.001). No correlation was found between the degree of enzymuria and either glomerular filtration rate or time since nephrectomy. The elevated activity of urinary enzymes in kidney donors may be related to increased metabolism by the renal tubular cells after contralateral nephrectomy. This study suggests that long-term compensatory hyperfiltration does not damage tubular cells, at least over this time scale.

Perchloric acid interference in enzymatic-fluorimetric-continuous-flow assay methods for measuring glucose, lactate, pyruvate, alanine, glycerol, and 3-hydroxybutyrate in blood.

Perchloric acid is commonly used to denature and precipitate proteins in samples before various metabolites are measured in tissue, blood, and other body fluids. However, perchloric acid can interfere in the analytical process, possibly by inhibiting the enzymes used. We have determined the effects of perchloric acid on measurements of glucose, lactate, pyruvate, alanine, glycerol, and 3-hydroxybutyrate in blood by enzymatic-fluorimetric-continuous-flow assays. There was a net increase or decrease in the apparent concentration of some of these metabolites when the perchloric acid concentration in the samples differed from that of the reference standards-some of these differences were due to the concentration of perchlorate ion and some to the pH of the acid extracts. The results show the need either to add a fixed amount of blood to perchloric acid or to neutralize and remove the perchlorate.

Separation of alkaline phosphatase isoenzymes from human kidney and comparison with alkaline phosphatases from other human tissues, urine, and Escherichia coli.

Human kidney isoenzymes of alkaline phosphatase (EC 3.1.3.1) after extraction with butan-1-ol were separated by ammonium sulfate precipitation, gel filtration, and chromatofocusing fractionation methods. The separation at each fractionation step was monitored by starch gel and equilibrium-gradient-pore electrophoresis, the latter technique also being used to determine molecular mass. The determined molecular mass (daltons) of alkaline phosphatase from human placenta was 132 000, from urine 95 000, and three isoenzymes from kidney were 195 000, 140 000, and 95 000, respectively. The mass of Escherichia coli alkaline phosphatase was 80 000 daltons, and that of human liver alkaline phosphatase was assumed to be 160 000 daltons. The urinary isoenzyme and the electrophoretically fastest migrating kidney isoenzyme were similar with regard to pH optima, charge, and molecular mass as well as response to L-phenylalanine, L-homoarginine, heat, and urea. Bacterial alkaline phosphatase could be distinguished from the alkaline phosphatases in human tissues and urine by differences in the response to changes in pH and several other physicochemical properties.

Diurnal hormone-metabolite profiles in hypothyroidism.

To investigate the influence of thyroid hormones on intermediary metabolism in man, hormone and metabolite profiles were obtained over a 12-h period of normal meals and activity in eight hypothyroid subjects before and during thyroxine replacement therapy, and in sixteen matched controls. The fasting blood glucose concentration and the mean 12-h blood glucose concentration were normal in hypothyroid subjects but the blood glucose response to breakfast was exaggerated. Fasting blood lactate and pyruvate levels were normal but post-prandial hyperlactataemia and hyperpyruvicaemia were found and mean 12 h values for lactate (hypothyroid 1.80 +/- 0.06 v. control 0.77 +/- 0.03 mmol/l, P less than 0.01) and pyruvate (0.10 +/- 0.01 v. 0.08 +/- 0.003 mmol/l, P less than 0.01) were elevated. Blood alanine concentrations were elevated only in the evening. Although plasma non-esterified fatty acid levels were normal, fasting blood glycerol levels were decreased (0.06 +/- 0.01 v 0.08 +/- 0.01 mmol/l, P less than 0.001) and this decrease persisted throughout the 12-h period. Blood total ketone body concentrations did not differ from controls, but, as for plasma NEFA and blood glycerol, the normal preprandial rise in concentration was absent. Serum insulin, glucagon and growth hormone concentrations did not differ from control values at any time. Six months of thyroxine (T4) treatment produced a rise in blood glycerol concentration (mean 12 h value during T4 therapy, 0.06 +/- 0.01; before T4 therapy, 0.04 +/- 0.005 mmol/l; P less than 0.01) but not to control values (0.08 +/- 0.01 mmol/l). Concentrations of glucose and other gluconeogenic precursors were unaltered by therapy but the insulin response to meals and the mean 12 h serum insulin concentration were increased.

Molecular weight of urinary alkaline phosphatase determined by equilibrium-gradient-pore electrophoresis.

A molecular weight of 95,000 for normal urinary alkaline phosphatase has been determined by equilibrium-gradient-pore electrophoresis. Several protein markers were used including alpha 2-macroglobulin, catalase, human liver alkaline phosphatase, serum transferrin and haemopexin.

Immunological identity of urinary alkaline phosphatase in health and disease.

Urinary alkaline phosphatase has been measured and an investigation has been undertaken involving starch gel electrophoresis after treatment of the enzyme with antisera raised against various human tissues. Urine was obtained from patients with bone and liver disease as well as pregnant women and normal persons. The enzyme level in urine in disease and pregnancy was raised above the normal but considerable overlap occurred. After electrophoresis the most usual finding was a single zone of activity although occasionally very minor zones were also present. Antiserum to kidney had the greatest affinity for the urinary enzyme. Decreasing affinity was found with intestinal and placental antiserum. No affinity was found with liver antiserum.

Human liver alkaline phosphatase purified by affinity chromatography, ultracentrifugation and polyacrylamide-gel electrophoresis.

A method is presented for the preparation of human liver alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1). The method gives a purification factor of 12.5 X 10(3) over the initial aq. butan-1-ol extract, a recovery of 6.0% and a specific activity for the preparation of 1450-1550 units/mg of protein, 1 unit being defined as the amount of enzyme catalysing the hydrolysis of 1mumol of p-nitrophenyl phosphate/min at 35 degrees C in 0.1 M-2-amino-2-methylpropan-1-ol/HCl buffer, pH 10.5, containing 10mM-p-nitrophenyl phosphate. Homogeneity was studied by ultracentrifugation, by immunoelectrophoresis and by polyacrylamide-gel electrophoresis. A single contaminating protein was present which was less than 5% of the total. Ultracentrifugation and equilibrium-gradient-pore electrophoresis techniques indicated a mol.wt. of 156000 and 160000 respectively. Equilibrium-gradient-pore electrophoresis indicated that the alkaline phosphatase molecule is possibly a dimer, comprising two subunits of about 80000 mol.wt. Amino acid analysis proved remarkably similar to that for alkaline phosphatase from other sources, regardless of species.

Alkaline phosphatase isoenzymes of human kidney and urine.

Kidney and urinary alkaline phosphatases have been studied by starch gel electrophoresis. Kidney extracts have shown individual variations and the patterns obtained with cortex and medulla have been clearly different. There are in urine and kidney extracts, phosphatases which share similar properties with regard to electrophoretic mobility, non-susceptibility to treatment with neuraminidase and inhibition by L-phenylalanine.