David J. Bates

Effects of birthweight and oxygen supplementation on lung function in late childhood in children of very low birth weight

Impaired respiratory function has been found frequently in ex‐premature children, but it is unclear which specific factors influence this impairment the most. The aim of this study was to determine the importance of the contributions of birth weight, gestational age, neonatal respiratory disease, and its treatment on subsequent childhood lung function at age 11 years in a cohort of children of very low birth weight (VLBW; ≤1,500 g). Detailed clinical histories were recorded, and lung function was measured in 60% (102 children) of surviving VLBW infants born 1981/1982, and compared with 82 matched control children (birth weight >2,000 g) of similar age.

Power Spectral Analysis of Heart Rate Variability in Children and Adolescents With IDDM

OBJECTIVE To investigate power spectral analysis (PSA) of heart rate variability (HRV) in children and adolescents with IDDM, its relationship with other measures of HRV and standard cardiovascular responses, and factors associated with reduced HVR. RESEARCH DESIGN AND METHODS A total of 130 subjects with IDDM aged 12.8 ± 3.2 years and 108 healthy control subjects were studied. Power spectra were analyzed from supine electrocardiograph (ECG) recordings by processing into consecutive R-R intervals and analysis using fast Fourier transformation. Standard cardiovascular responses to deep breathing and standing were performed. RESULTS IDDM subjects had a reduction in total power including both low-frequency (0.05–0.14 Hz; P = 0.0001) and high-frequency (0.14–0.40 Hz; P = 0.0002) components. These changes were seen from diagnosis. Other measures of HRV, coefficient of variation (CV) and standard deviation (SD) of mean resting heart rate, were also significantly lower in IDDM. All 20 (15%) of the 130 IDDM subjects with total power < the 5th percentile in control subjects also had reduced HRV when measured by CV of heart rate. There was an independent relationship between age and the high-frequency component in IDDM subjects and control subjects. Total power correlated with mean heart rate (r = −0.56; P < 0.0001), CV of heart rate (r = 0.90; P < 0.00001), SD of heart rate (r = 0.91; P < 0.00001), heart rate response to deep breathing (r = 0.45; P < 0.0001), and duration in IDDM subjects. There was no correlation with short-term or long-term metabolic control. Retesting of 27 subjects showed a variability in total power and its components comparable to other measures of HRV and standard heart rate responses. CONCLUSIONS Changes in HRV are a sensitive and reproducible measure of early autonomic dysfunction in childhood. In this age-group, PSA appears no more sensitive a measure of reduced HRV than other closely correlated measures of HRV.

Progression of borderline increases in albuminuria in adolescents with insulin-dependent diabetes mellitus

We aimed to determine the natural history of borderline increases in albuminuria in adolescents with insulin‐dependent (Type 1) diabetes mellitus (IDDM) and factors which are associated with progression to persistent microalbuminura. Fifty‐five normotensive adolescents with IDDM and intermittent microalbuminura (overnight albumin excretion ratte of 20–200 μg min−1 on one of three consecutive timed collections, n  = 29) or borderline albuminura (mean overnight albumin excretion rate of 7.2–20 μg min−1 on one of three consecutive timed collections, n  = 30) were followed prospectively at 3 monthly intervals. The endpoint was persistent microalbuminuria defined as a minimum of three of four consecutive overnight albumin excretion rates of greater than 20 μg min−1 . One hundred and forty‐two adolescents with IDDM and normoalbuminura were also followed prospectively. Fifteen of the 59 patients (25.4 %) with intermittent (9/29) or borderline (6/30) albuminura progressed to persistent microalbuminura (progressors) over 28 (15–50) months [median (range)] in comparison with two of the 142 patients with normoalbuminuria at entry (relative risk =12.6; p  =0.001). Progressors to persistent microalbuminura were pubertal and had higher systolic (p  = 0.02) and diastolic (p  = 0.02) blood pressure, and HbAlc (p  = 0.004) than non‐progressors. All patients remained normotensive. Glomerular filtration rate, apolipoproteins, dietary phosphorus, protein and sodium intakes, and prevalence of smoking did not differ between progressors and non‐progressors. Total renin was higher in the diabetic patients without a difference between progressors and non‐progressors. In conclusion there is a relatively high rate of progression to persistent microalbuminuria in pubertal adolescents with borderline increases in albuminura and duration greater than 3 years. These patients require attention to minimize associated factors of poor metabolic control and higher blood pressure in the development of incipient nephropathy. © 1997 John Wiley & Sons, Ltd.

Association of Lipoprotein(a) With Puberty in IDDM

OBJECTIVE— To determine serum lipoprotein(a) in a large sample of IDDM and control children and to examine a possible association with puberty. RESEARCH DESIGN AND METHODS—Serum lipoprotein(a), apoB-100, and apoA-I were measured under identical conditions in 170 Caucasian children with IDDM aged 12.3 ± 3.59 yr and 233 Caucasian control children aged 13.6 ± 1.12 yr. Patients with persistent microalbuminuria were excluded. Lipoprotein(a), apoB-100, and apoA-I were measured by nephelometry using a specific monoclonal antibody. Pubertal assessment was performed using Tanner staging and testicular volume measurement. RESULTS— Lipoprotein(a) was higher in the IDDM than control group (geometric mean 237 mg/L, 25–75th percentile 134–465 vs. 172 [99–316] mg/L, P = 0.0008). When analyzed according to pubertal stage, only pubertal and postpubertal patients had higher levels than control subjects (265 [148–560] vs. 174 [101–320] mg/L, P = 0.0001), with prepubertal patients showing no difference. Pubertal and postpubertal patients showed both higher lipoprotein(a) (P = 0.01) levels and higher albumin excretion rates (P = 0.02) than prepubertal patients, correcting for the other variable. Lipoprotein(a) was not related to HbA1c, albumin excretion rate, duration, age, sex, mean arterial pressure, or a family history of premature coronary artery disease in the IDDM group. Lipoprotein(a) was not higher in patients with overnight albumin excretion rate above the 95th percentile but below the microalbuminuric range. ApoB-100 did not differ between IDDM and control children. ApoA-I was significantly lower in the IDDM group (1.04 [0.94–1.17] vs. 1.21 [1.10–1.31] g/L; P < 0.0001). CONCLUSIONS— Pubertal and postpubertal IDDM patients have higher serum lipoprotein(a) than Caucasian control subjects. Our findings suggest a rise in lipoprotein(a) may occur during puberty in IDDM. Longitudinal studies are required to clarify the relationship between lipoprotein(a), albumin excretion rate, and puberty.

The metabolism of cyclophosphamide by isolated rat hepatocytes

Abstract The metabolism of cyclophosphamide was studied in vitro using isolated rat hepatocytes and mass spectrometry. The major product of primary oxidative metabolism in hepatocytes from phenobarbital treated rats was 4-hydroxycyclophosphamide, isolated as the O-ethyl derivatives, but dechloroethylation was also a substantial pathway. 4-Hydroxycyclophosphamide was converted mainly into carboxy phosphamide and the formation of 4-ketocyclophosphamide was a minor pathway. Evidence is presented that under certain conditions a substantial amount of an O-glucuronide of 4-hydroxycyclophosphamide was formed. The pattern of metabolism in hepatocytes otherwise resembled qualitatively that observed previously in vitro using subcellular fractions and in vivo, but quantitative differences were found. The metabolism of cyclophosphamide by hepatocytes resembles more closely that in vivo than does the metabolism in subcellular fractions, and hepatocytes should be the preferred in vitro system for studying the metabolism of anti-tumour agents.

Inversely related oscillations in the contents of cyclic GMP and the total adenine nucleotides in steady-state perfused rat hearts

While investigating amino acid incorporation into protein in Langendorf perfused hearts we made the surprising observation that the tissue contents of cyclic AMP and cyclic GMP showed very significant variations when measured at time points 20 rain apart during a 2 h perfusion period [1]. Despite these large temp oral variations in the cyclic nucleotides, the heart preparation is apparently in a steady-state for up to 2 h as judged by oxygen uptake, glucose utilization, nicotinamide nucleotide ratio, glycogen turnover, lactate turnover, alanine output [2,3] protein-synthesis rate [1,4], beat rate and contraction amplitude [5]. It had been suggested that cellular feedback-regulation systems might give rise to stable oscillations on which metabolic steady-states could rely [6] and moreover oscillations which appeared to be related to glycolytic oscillations had been observed in the concentrations of the adenine nucleotides and IMP in particle-free rat skeletal-muscle extracts [7]. The possibility that such oscillatory phenomena were responsible for the cyclic nucleotide variations was investigated by measuring in both hearts and perfusate all purine nucleotides, nucleosides and their degradation products at selected time points over 80 min of perfusion. Highly significant differences were found in the contents of ATP, ADP, GTP, cyclic AMP, cyclic GMP in the ATP/ADP ratio and in the sum of the adenine nucleotides at time points 10 or 20 min apart [5]. The ATP/ADP ratio and the contents of GTP and cyclic AMP showed an identical pattern of variation which was distinct from that of the total adenine nucleotide content. The very large nett increases and decreases in the total adenine nucleotides extracted could neither be explained as operation of Lowensteins purine nucleotide cycle [7] nor as metabolism of any known nucleotide precursor, derivative or polymer and it was suggested that an unsuspected substantial storage form of purine nucleotide might exist in mammalian heart [5]. This paper reports that when hearts are sampled more frequently very regular statistically significant temporal variations in total adenine nucleotide content are discerned. Furthermore the changing adenine nucleotide content is mirrored by inverse alterations in the cyclic GMP content. Since the cyclic GMP content also appears to respond to the non-synchronous variations in total guanine nucleotide, it is argued that guanylate cyclase activity may be regulated by the level of free adenine nucleotide in the cell.

In perfused rat hearts ischaemia promotes the reversible conversion of appreciable quantities of soluble adenine nucleotides to a stable trichloroacetic acid-precipitable form

Radioactivity from [14C] adenosine was linearly incorporated into tissue nucleotides in perfused rat hearts. All the TCA-extractable 14C was confined to the purine nucleoside phosphates for up to 1 h of perfusion. Radioactivity was also incorporated linearly into the TCA-insoluble fraction, which by 40 min accounted for 24% of the tissue 14 C. Estimates based on precursor specific radioactivity suggest that at least 0.6 micro mol/g of the mononucleotide is in this stable insoluble form. Following 2 min total ischaemia, the tissue nucleotide content and soluble radioactivity decreased while the insoluble radioactivity showed a corresponding increase to account now for 35% of the tissue radiolabel. This redistribution was rapidly reversed by post-ischaemic reperfusion. A possible function for the rapid reversible sequestration of adenine nucleotides in ischaemia is proposed.

Blot and culture analysis of neuronotrophic factors in nerve regeneration chamber fluids

The fluid accumulating in silicone nerve regeneration chambers implanted between the cut ends of rat sciatic nerve contains neuronotrophic activities towards embryonic chick ciliary and sympathetic neurons. The blot and culture technique of Carnow et al. was used to determine if part of the neuronotrophic activities is due to ciliary neuronotrophic factor, which supports the survival of both types of neurons in vitro. The technique involves separating the fluid proteins by SDS-polyacrylamide gel electrophoresis, Western transfer, and then culturing of purified neurons on the nitrocellulose blots. After 24 hr surviving neurons are restricted to regions of the blot where neuronotrophic factor is present. Analysis of 1 and 2 day fluids showed that a multitude of factors are present, particularly in the 19–30 kD molecular weight range, with discrete peaks of activity at molecular weights consistent with those reported for ciliary neuronotrophic factor. There were several other peaks of activity present in the fluids in addition to these.

Ribonuclease activities in rat sympathetic ganglia: Evidence for the presence of an endogenous inhibitor of alkaline ribonuclease

Using3H-labeled rat brain mature RNA as substrate, substantial ribonuclease activity was detected in homogenates of rat superior cervical ganglia with acidic (pH 5.5) and neutral (pH 7.0-7.5) optima. Very little activity could be measured at greater than pH 8. The acidic and neutral activities differed in the optimal conditions required for assay, and showed differential sensitivity to the sulfhydryl blocking agent, N-ethylmaleimide. Only the neutral activity was stimulated, optimally by 2 mM N-ethylmaleimide, and the magnitude of stimulation indicated that the contributing ribonucleases exist largely in a latent form in the ganglion. Ribonucleases in other tissues with neutral pH dependence, known usually as “alkaline” ribonucleases, are subject to an N-ethylmaleimide-sensitive endogenous inhibitor protein. The existence of a similar inhibitor in rat superior cervical ganglia was indicated by the latency of neutral ribonuclease activity and confirmed by observing the effect of a soluble fraction from the ganglia on the activity of pancreatic ribonuclease A.

Multiple neurotrophic factors including NGF-like activity in nerve regeneration chamber fluids ☆

Silicone nerve regeneration chambers were implanted between the cut ends of the sciatic nerve of adult rats. Neurotrophic activities in cell-free fluids collected from the chambers were determined using bioassays for survival of embryonic chick ciliary and sympathetic neurons in culture. Separation by molecular exclusion HPLC of the components of fluids collected 1, 2 or 3 days after implantation revealed the presence of a multitude of neurotrophic factors differing in their molecular weights, specificity towards the two types of neurons, and time course. Antiserum to nerve growth factor partially blocked sympathetic activity of fluids collected at 1 day. Affinity purified antibody was also effective and completely eliminated bioactivity of HPLC fractions corresponding to the molecular weight of nerve growth factor. The presence in the fluids of 13-18 and 20-32 kD components active towards ciliary neurons is consistent with the release of fibroblast growth factor and ciliary neurotrophic factor respectively. The stimulation of sympathetic neurons by the 13-18 kD material, and also by 4-6 and 7-11 kD components cannot be entirely accounted for by known factors. This study demonstrates that a number of neurotrophic factors, which differ in their specificity towards sympathetic and parasympathetic neurons, are made available to the region of axonal regrowth over the first few days of regeneration. Contrary to earlier reports, nerve growth factor-like activity was shown to be present in nerve regeneration chambers.