J. M. Bridges

Erythropoietin production in kidney tubular cells

The erythropoietin gene has been cloned in three mammalian species including man and recombinant erythropoietin is now used to treat the anaemia of chronic renal failure. Despite the isolation of the gene the precise cellular location of erythropoietin synthesis remains controversial. We present studies which demonstrate erythropoietin production by kidney tubular cells. Erythropoietin gene expression (messenger RNA) was detected by in situ hybridization using an oligonucleotide gene probe and the translated protein product by immunohistochemistry employing antibodies raised to pure recombinant DNA derived erythropoietin.

Comparison of the mouse spleen cell assay and a radioimmunoassay for the measurement of serum erythropoietin

The mouse spleen cell assay (MSCA) has been compared with a radioimmunoassay for the measurement of serum erythropoietin (Ep). In 20 normal subjects the serum values ranged from 15 to 73 mU/ml for the MSCA compared with 5‐30 mU/ml for the RIA. For normal sera there was no correlation between the results of the two assays. In 37 patients with anaemias of differing aetiologies and at various stages of treatment values ranged from 10 to 3645 mU/ml for the MSCA and 13‐10 000 mU/ml for the RIA. Although patient values from the two assays were highly correlated (r=0.98, P < 0.001), the MSCA results were generally lower. These discrepancies can be largely accounted for by two factors. Firstly the MSCA is sensitive to non‐specific matrix effects. Secondly, heat inactivation of serum, a prerequisite for the MSCA, but not for the RIA, destroys a variable and unpredictable proportion of the Ep in the test sera leading to an underestimation of Ep in the MSCA. We conclude that the RIA is more reliable than the MSCA which, in its present form, cannot be recommended for the accurate measurement of serum erythropoietin.

Renal function in the elimination of oral melphalan in patients with multiple myeloma

SummaryPharmacokinetic studies in 11 patients with multiple myeloma were undertaken on the first and last days of one course of chemotherapy. The drug was administered PO in single doses of 6–14 mg daily. Melphalan concentrations were determined by high-performance liquid chromatography. The interpatient variability of pharmacokinetic parameters noted by other authors was observed. Regression analysis showed a significant positive correlation between the elimination rate constant for melphalan and renal function (P=0.003). The form of the line which describes the overall elimination rate constant for melphalan is given by the equation: Kel=5.67×10-3+[4.90×10-5xGFR]. There was also a significant negative correlation between renal function and the area under the plasma melphalan concentration/time curve (P=0.006). In vitro stability studies of melphalan in plasma at 37°C and pharmacokinetic data suggest that hydrolysis and renal clearance are the major mechanisms of melphalan elimination. This work shows quantitatively the relationship between renal function and drug elimination and how the data may be used in predicting melphalan half-life from creatinine clearance.

Enumeration of absolute numbers of T lymphocyte subsets in B-chronic lymphocytic leukaemia using an immunoperoxidase technique: relation to clinical stage

Summary. An immunoperoxidase technique has been used to identify and enumerate helper and suppressor T‐cell subsets, as defined by reactivity with Coulter T4 and OKT8 monoclonal antibodies in 54 patients with B chronic lymphocytic leukaemia (B‐CLL) and in the same number of matched controls. The ratio of T4+ to T8+ cells was significantly reduced in the B‐CLL group as a whole (P<0.001) and in each stage of the three clinical staging systems. There was an increase in the median absolute level of T8+ cells in the whole CLL group (P<0.001). However, subdivision of the CLL group by clinical staging systems revealed a large group (28 patients) in which median T8+ cell levels were not raised and median T4+ cell levels were low (P<0.001). There was no significant decrease in T4:T8+ ratio, increase in T8+ cells or decrease in T4+ cells with progression of clinical stage. Absolute numbers of E+ cells were significantly raised in all staging systems as were E+ T4− T8− cells (P< 0.001). A significant alteration in either of these populations with progression of clinical stage was not present.

Can food affect the bioavailability of chlorambucil in patients with haematological malignancies

SummaryPharmacokinetic studies in ten patients with haematological disorders were undertaken on the first and second days of one course of chemotherapy. Patients received chlorambucil under fasting and non-fasting conditions. Plasma concentrations of chlorambucil were determined by a reversed-phase high-performance liquid chromatography assay. Statistical analysis by the Wilcoxon signed rank test for non-parametric data indicated that food caused a significant reduction in peak plasma levels (P<0.01), elimination rate constants (P<0.01) and area under the plasma chlorambucil/time curve (P=0.01). Food was also found to prolong the time taken to attain peak plasma levels (P<0.01). Regression analysis of renal function with elimination rate constants showed that chlorambucil elimination was independent of renal function (n=8; r=-0.007; P=0.72). In view of these results we suggest that chlorambucil is given on an empty stomach.

The proliferative activity of B-chronic lymphocytic leukaemia lymphocytes prior to and after stimulation with TPA and PHA

The proliferative activity of B-CLL lymphocytes from 10 patients was investigated both prior to and after stimulation with TPA and PHA. The analysis of cell cycle-associated features such as BrdU incorporation and the expression of the nuclear proliferation-associated antigen, Ki-67, together with the phenotypic profile of the cells, was performed using double colour immunofluorescent methods. The unstimulated B-CLL cells represented a homogeneous population with the same cell cycle position (G0) as resting peripheral blood lymphocytes. After TPA stimulation 22.7% of the lymphocytes were found in G1, 9.4% in S + G2/M and 13.4% in post-M. PHA stimulation induced a greater proportion of cells in G1, i.e. 35% and 17.8% into S + G2/M and 13.4% into post-M. Double colour immunofluorescence was able to demonstrate that in TPA cultures the majority of the stimulated lymphocytes originated from the malignant clone. Evidence of B-CLL lymphocyte proliferation using double colour labelling with BrdU and Ig kappa and/or Ig lambda showed that a small minority of B-CLL lymphocytes were stimulated into S + G2/M phases of the cell cycle. PHA was also capable of inducing a small proportion of B-CLL cells into mitosis although this proportion of cells was smaller compared to the TPA-stimulated lymphocytes.

Three Pyruvate Kinase Variants with Increased Affinity for PEP

Summary. Three variants of pyruvate kinase are described which have marked reduction of activity associated with severe non‐spherocytic haemolytic anaemia. Each variant shows a reduced K0.5 PEP (the value of the intercept of the abscissa on the Hill plot) and reduced Hill coefficient; FDP activation and ATP inhibition are less than normal and utilization of GDP is increased. The variants are slightly less inhibited by 2,3DPG than controls but require more FDP to relieve this inhibition. Cases 1 and 2 have decreased thermostability but case 3 is normal. The mutant enzymes are further distinguished by their affinity for FDP. Their kinetic and physicochemical properties are compared with other known cases with high affinity for PEP and discussed in terms of a RT model for allosteric enzymes.

Increased plasma glycosidase and protease activity in uraemia: possible role in the aetiology of the anaemia of chronic renal failure

We have measured plasma N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30) and neuraminidase (EC 3.2.1.18) activities as markers of glycosidase activity and immunoreactive trypsin (EC 3.4.21.4) levels as a marker of proteolytic potential in the plasma of normal and uraemic subjects. The levels of all of these enzymes are significantly elevated in the plasma of uraemic subjects when compared to normal. We have postulated that the combined attack of glycosidases and proteases on erythropoietin will lead to fragmentation of this glycoprotein hormone with loss of activity. This may be a major contributory cause to the anaemia of chronic renal failure.

Serum erythropoietic activity in acute anemia--an animal model

Serum erythropoietic activity and reticulocyte response to anemia were investigated using a rabbit model. In hemolytic anemia, induced by injections of phenylhydrazine on Day 0 the hemoglobin reached a nadir (mean, 6.23 g/dl) on Day 4 when SEA was maximal (mean, 765 mU/ml). In animals venesected on Day 0 and Day 1 to produce anemia of equal severity, the SEA was maximal (mean 235 mU/ml) on Day 2. In both groups the reticulocyte response peaked on Day 7--at 34% for the hemolytic group and 21% for the venesected group. The 2,3-diphosphoglycerate, measured on Day 4, was significantly reduced in the PHZ-treated group. In the venesected group the 2,3-DPG increased between Day 0 and Day 4. There were no concurrent changes in acid-base balance. These results imply that the degree of anemia is only one of the factors which influence the level of circulating SEA.

Lymphocytes from patients receiving lithium do not inhibit CFU-C growth

Summary. Lymphocyte subset levels and function were examined in 12 patients on lithium therapy and in 11 healthy hospital personnel. Co‐culture of allogeneic human bone marrow cells with monocyte‐depleted lymphocyte preparations revealed that CFU‐C formation was significantly reduced (mean 43% inhibition) in the presence of normal lymphocytes but not with the patients’ lymphocytes (< 5% inhibition). This did not reflect numerical changes in lymphocyte subsets, since these were similar for control and lithium subjects. T colony formation was significantly depressed in the patient group (P <0.05), whereas B colony numbers were similar in both groups (P > 0.1). The possible role of HLA‐incompatability affecting CFU‐C growth was investigated in co‐culture experiments, using lymphocytes from HLA‐identical twins, one of whom was receiving lithium. In four separate co‐culture experiments, the inhibitory effect was shown with lymphocytes from the non‐lithium twin but was not demonstrated by the lithium subject. Addition of lithium in vitro to co‐cultures of normal marrow and lymphocytes was found to negate the inhibitory phenomenon in a dose‐related manner. It is postulated that granulocytosis induced by the administration of lithium may be a manifestation of changes in a lymphocytic control system.