Germ cell damage and Leydig cell insufficiency in recipients of nonmyeloablative transplantation for haematological malignancies.

Most bone marrow transplant recipients are infertile due to reversible or irreversible testicular failure. However, little is known about the gonadotoxic potential of the newly introduced nonmyeloablative transplants. We undertook a 24-month longitudinal study in a cohort of 32 recipients of nonmyeloablative transplantation to test whether the combined regimen of fludarabine, melphalan and CAMPATH-1H can induce damage to germ cell (GC) and Leydig cell (LC) compartments. Testicular function was assessed immediately prior to transplantation and at four time points post-transplant to compare hormonal levels before and after the procedure. Two other groups treated with BEAM- and TBI-related regimes were also included in the study group for comparative purposes. GC function was assessed by measuring basal serum follicle stimulating hormone (FSH). LC function was assessed by measuring basal luteinising hormone (LH) and testosterone (T) levels. LC reserve was assessed by measuring the T/LH ratio. As a group, patients who received a non myeloablative transplant sustained severe damage to the GC compartment, as evident from a substantial elevation in the FSH level post-transplant (12 IU/l vs 18.4 IU/l, P<0.001). Similar to the GC injury, patients as a group sustained significant damage to the LC compartment following the transplant (5.4 IU/l vs 9.6 IU/l, P<0.001). In general, patients had reduced LC reserve post-BMT, as evident from a diminished T/LH ratio (2.6 pretransplant vs 1.6 post-transplant P=0.05). Patients who received a nonmyeloablative transplant had a similar effect on the GC and LC compartments compared to those who had a BEAM autograft. On the other hand, patients who received a TBI-based transplant sustained more damage to their GC and LC compartments compared to those who received a nonmyeloblative transplant; however, this was not statistically significant (P=0.09). Our data suggest that this type of regimen is potentially gonadotoxic and consideration should be given to fertility counselling and testosterone replacement therapy post-transplant.

Relationship between myoinositol influx and lipids in diabetic neuropathy.

Diabetic neuropathy is associated with abnormalities in lipid metabolism and has been postulated to be associated with abnormal myoinositol metabolism. Leucocyte myoinositol influx was measured using a triple isotope method in long-standing type 1 (insulin-dependent) diabetic patients with and without diabetic neuropathy and in a group of matched controls. No differences in fasting lipid, glucose concentrations or glycated haemoglobin were found in the diabetic groups. Myoinositol influx was significantly but negatively correlated with the serum very low density lipoprotein (VLDL) cholesterol concentration in patients with and without neuropathy but not in the controls. The VLDL cholesterol concentration also correlated negatively with the transporter Km (Rs = -0.87, P < 0.005, neuropathic group only) and Vmax (Rs = -0.93, P < 0.001, neuropathic group; Rs = -0.59, P < 0.05, non-neuropathic group). Myoinositol influx correlated with the glycated haemoglobin only in the diabetic patients without neuropathy. The lipid relationships in diabetic subjects were independent of glucose control, which suggests that myoinositol influx mechanisms represent another transporter affected by intracellular lipid metabolism. The control of VLDL metabolism by fibrates could offer a method for reducing the progression of diabetic neuropathy.

Effects of the beta-adrenergic agonist, ritodrine, and insulin on plasma potassium concentrations in fetal lambs.

Prolonged infusion of the beta 2-adrenergic agonist, ritodrine, into sheep during late pregnancy decreased maternal plasma K+ from 3.6 to 2.5 mmol l-1 during the first 6-8 h of infusion, as it does during tocolysis in women. This decrease was not accompanied by significant change in fetal plasma K+ concentration. Ritodrine infusion (1-3.5 micrograms kg-1 min-1) directly into the fetus also did not decrease fetal plasma K+ significantly. In contrast, insulin (2.5 mU kg-1 min-1), infused together with glucose (3.6 mg kg-1 min-1) directly into the fetus decreased fetal plasma K+ concentration by 0.8 mmol l-1 within 1 h. The results suggest immaturity in beta 2-adrenergic receptor regulation of electrogenic K+ uptake by muscle in fetal lambs.

Abnormal myoinositol influx in human leucocytes in diabetes but not specifically in diabetic neuropathy.

Abnormal myoinositol metabolism has been implicated as a contributor to the development of diabetic neuropathy. Furthermore, in vitro glucose inhibits animal and human myoinositol transporters. To investigate whether myoinositol transport is abnormal in diabetic subjects with and without neuropathy, we used a triple-isotope technique to measure [14C]myoinositol uptake in leucocytes from 23 insulin-dependent diabetic subjects and 13 matched nondiabetic subjects. All subjects with diabetes underwent neurophysiological studies, and subjects without neuropathy were compared with those with various degrees of neuropathy. The relationship between glycemia and flux was also studied. Diabetic subjects had similar intracellular and plasma myoinositol concentrations but had higher rates of uptake of myoinositol over the extracellular concentrations of myoinositol studied. Although the derived Km, Vmax, and passive components were not significantly different, the Vmax:Km ratio was significantly higher in diabetic subjects compared with nondiabetic subjects (0.25 [0.17-0.32] vs. 0.16 [0.13-0.19], respectively (P = 0.006). In diabetic subjects, the rate of myoinositol uptake correlated with HbA1c, particularly at 3 microM extracellular myoinositol where active uptake was a high proportion of the total influx (P less than 0.005). No difference in myoinositol uptake was found among diabetic subjects with various degrees of neuropathy. We conclude that although myoinositol transport is abnormal in diabetes, it is not specifically abnormal in diabetic neuropathy. Prolonged hyperglycemia is associated with higher myoinositol flux.

Stimulation of the human leucocyte Na+/H+ antiport by D-glucose is mediated by protein kinase C.

Leucocyte Na+/H+ antiport activity is elevated in patients with essential hypertension and Type 1 diabetes with nephropathy. To examine the effects of hyperglycemia on the Na+/H+ antiport, normal leucocytes were incubated with 25 mmol l-1 D-glucose, L-glucose or glucose-6-phosphate for two days. Leucocyte Na+/H+ antiport activity was measured by a novel double ionophore fluorimetric method for controlling intracellular pH. Only incubation with D-glucose led to an increase in Na+/H+ antiport activity of about 31%. This effect was not due to non-enzymic glycation since glucose-6-phosphate, which glycates proteins faster than D-glucose, caused no significant difference in antiport activity. Also, osmotic effects could be excluded. Staurosporine (10 nmol l-1), a specific inhibitor of protein kinase C, prevented the rise in antiport activity due to incubation with D-glucose. As hyperglycaemia is known to increase protein kinase C activity, elevation of this kinase may be one mechanism for activation of the Na+/H+ antiport in Type 1 diabetes.

Leucocyte Na+/H+ antiport activity in type 1 (insulin-dependent) diabetic patients with nephropathy.

The development of proteinuria in Type 1 (insulin-dependent) diabetic patients may depend on predisposition to essential hypertension in addition to poor glycaemic control. Previous work has shown increased leucocyte Na+/H+ antiport activity in essential hypertension and increased erythrocyte Li+/Na+ exchange in Type 1 diabetic patients with proteinuria. To test whether susceptibility to nephropathy in Type 1 diabetes was linked to abnormalities of leucocyte Na+/H+ antiport activity, we measured the intracellular pH and kinetics of the Na+/H+ antiport in 19 Type 1 diabetic subjects with, and 15 diabetic subjects without albuminuria and compared them to 25 matched normal control subjects. Intracellular pH (mean +/- SD 7.59 +/- 0.14) and maximal transport capacity of the antiport (Vmax 87.7 +/- 24.9 mmol.1-1.min-1) were higher in diabetic subjects with albuminuria compared to normotensive control subjects (pH 7.44 +/- 0.09; Vmax 55.6 +/- 10.3 mmol.l-1.min-1; p less than 0.001 for both), similar to the defect described in essential hypertension. These differences were not seen in diabetic subjects with normal urinary albumin/creatinine ratios (pH 7.46 +/- 0.09; Vmax 61.0 +/- 13.6 mmol.l-1.min-1). Buffering characteristics of the leucocytes at different pH in the Type 1 diabetic subjects with albuminuria differed from normal control subjects and diabetic subjects with normal urinary albumin/creatinine ratios. We conclude that increased leucocyte Na+/H+ antiport activity, a known marker of essential hypertension, is usually associated with nephropathy in Type 1 diabetes.

Effect of protein kinase C modulators on the leucocyte Na+/H+ antiport in type 1 (insulin-dependent) diabetic subjects with albuminuria.

It is uncertain why only one third of Type 1 (insulin-dependent) diabetic patients develop nephropathy. One suggestion is the inheritance of a predisposition to essential hypertension. We have previously found elevated Na+/H+ antiport activity and a raised intracellular pH in leucocytes from hypertensive and Type 1 diabetic subjects with albuminuria using a novel double ionophore fluorimetric technique. These changes are not found in Type 1 diabetic subjects without albuminuria. We wished to test the effect of a protein kinase C inhibitor staurosporine (100 nmol/l) on the elevated antiport activity, and the degree of stimulation achieved by exogenous diacyl glycerol. Raised leucocyte Na+/H+ antiport activity of Type 1 diabetic subjects with albuminuria (73.8 +/- 17.2 mmol.l-1.min-1) was restored to normal levels with staurosporine (54.9 +/- 17.9 mmol.l-1.min-1, p less than 0.001). The leucocyte Na+/H+ antiport activity of diabetic subjects without albuminuria fell significantly also with staurosporine but to a lesser extent (57.3 +/- 11.6 to 50.0 +/- 12.8 mmol/l, p less than 0.003). In contrast, leucocytes from normal control subjects showed no change in antiport activity with staurosporine (54.3 +/- 8.5 to 52.6 +/- 10.4 mmol.l-1.min-1). Dioctanoyl glycerol stimulated the leucocyte Na+/H+ antiport in normal subjects and diabetic patients without albuminuria, with significantly less stimulation in diabetic patients with albuminuria. We conclude that reversal by staurosporine of the elevated Na+/H+ antiport activity in Type 1 diabetic subjects with albuminuria could indicate a role for protein kinase C in activating the antiport. This hypothesis is supported by the reduced stimulation of the antiport by dioctanoyl glycerol in this group of patients.

myo-inositol influx into human leucocytes: methods of measurement and the effect of glucose.

1. Low intracellular concentrations of myo-inositol in diabetic cells may contribute to the development of tissue damage. The cause of these low levels is unknown, but inhibition of a putative myo-inositol transporter by high concentrations of glucose has been proposed. We have developed a triple-isotope method for estimating myo-inositol influx into human leucocytes and so investigated both the kinetics of this uptake in normal volunteers and the effect of glucose upon it. 2. Uptake was composed of a passive component with a rate constant of 2.4 +/- 0.3 X 10(-2) min-1 and a saturable component with a Km of 61 +/- 23 mumol/l and Vmax of 11.3 +/- 4.5 X 10(-4) mmol min-1 l-1. Ouabain and low extracellular concentrations of sodium partly inhibited influx. Uptake was predominantly into the cytosolic fraction of the cell with 12% entering the membrane-associated fraction at both 5 and 10 min. 3. myo-Inositol influx was significantly inhibited by both D- and L-glucose but not by sucrose. Neither cytochalasin B nor ethyl isopropyl amiloride significantly inhibited uptake. 4. It is concluded that a myo-inositol transporter exists in human leucocytes which is similar to that found in other species and tissues. Our technique allows myo-inositol influx in diabetic subjects to be related to varying glycaemic control and tissue damage.

Leucocyte intracellular pH and Na+/H+ antiport activity in human hypertension.

Hypertension is associated with thickening of the wall of resistance vessels, but the cellular or genetic basis of this is unclear. Cell proliferation and intracellular alkalinization via increased Na+/H+ exchange are linked in the response of tissues to growth factors. To define a possible cellular basis for vascular medial thickening in hypertension, we studied leucocyte intracellular pH, buffering power and Na+/H+ antiport activity in 17 hypertensive and 17 age-, sex- and weight-matched normotensive subjects. The cells from hypertensive subjects were significantly more alkaline [median (range): 7.49 (7.26-7.95) versus 7.39 (7.25-7.53); P less than 0.01], and had a lower buffering power [8.95 (3.05-17.98) versus 12.57 (7.44-19.95) mmol/l per pH unit; P less than 0.02] than those from normotensive subjects. Moreover, the activity of the Na+/H+ antiport was higher when cells were acid-loaded to an intracellular pH of 6.7. The presence of a similar increased activity in vascular smooth muscle cells may be associated with increased cellular proliferation resulting in a thickened media or increased vascular smooth muscle contractility.

Altered stoichiometry of the human leucocyte Na+/H+ antiport with decreasing pH.

The Na+/H+ antiport is an important regulator of cellular volume, pH and Na+ concentration in mammalian cells. The stoichiometry of this antiporter has previously been shown to be a 1:1 exchange of internal H+ for external Na+. We have investigated this stoichiometry in human leucocytes by using a novel intracellular pH-clamping technique and measuring 22Na+ influx and H+ efflux in the same cells. As internal pH was lowered, the stoichiometry of H+/Na+ exchange rose to a mean +/- S.D. of 2.23 +/- 0.69. This mechanism allows a higher H+ efflux in the face of intracellular acid stress without causing excessive intracellular Na+ overload.

Photoperiod: an important regulator of plasma prolactin concentration in fetal lambs during late gestation.

Measurement of prolactin (PRL) at 120-140 days gestation in chronically-cannulated fetal lambs and their mothers during hormonally-extended breeding seasons shows that photoperiod is a major determinant of PRL secretion in the fetal lamb as well as in its mother. About 80% of the variance between fetuses in plasma PRL concentration is associated with variation in ambient photoperiod. The magnitude of this effect far exceeds the range of previously reported diurnal or ontogenetic changes in PRL in the fetal lamb.

The gastrointestinal absorption of organically bound forms of plutonium in fed and fasted hamsters.

Values of about 0.005-0.01 per cent were obtained for the absorption in fed hamsters of plutonium ingested as Pu4+ citrate, isocitrate, phytate and malate complexes and Pu3+ ascorbate compared with about 0.003-0.004 per cent for Pu4+ nitrate. Replacing drinking water with tea did not affect the result for Pu4+ nitrate. Fasting hamsters for 8 h before the administration of plutonium citrate increased absorption to 0.1-0.2 per cent. An extra period of fasting for 4 h after administration did not lead to a further increase in absorption. Similar values were also obtained when plutonium citrate was administered after a 24 h fast, followed either by immediate access to food or a further 4 h fast. In hamsters fasted for 24 h before administration of either Pu3+ ascorbate or Pu4+ nitrate, about 6-7 per cent of the ingested plutonium was retained in the gastrointestinal tract after one week. At three weeks after ingestion of Pu3+ ascorbate, gastrointestinal retention had fallen 100-fold without an increase in absorption.