21-hydroxylase genotyping in Australasian patients with congenital adrenal hyperplasia.

Mutations in CYP21 (21-hydroxylase) lead to congenital adrenal hyperplasia (CAH). We genotyped 26 probands with CAH by PCR-sequencing the entire CYP21 gene. 25/26 had homozygous or compound heterozygous mutations. The frequencies of mutations were similar to other populations with deletion/hybrid, I2 G splice and I172N the most common. Five patients with a I172N allele predicting simple-virilising CAH had a salt-wasting phenotype. Two other probands also had a more severe phenotype than predicted by genotype. Two families had both non-classic and salt-wasting phenotypes arising from combinations of three deleterious alleles. Two novel CYP21 alleles were detected: D106N and a large deletion encompassing CYP21 and adjacent pseudogene. Two rare CYP21 alleles were also found. Three of these four novel/rare alleles were only detected as a result of sequencing the entire CYP21 gene. Entire CYP21 sequencing will increase the number of mutations detected in CAH, and in combination with functional studies should contribute a greater understanding of phenotype-genotype correlations.

The effect of breastmilk and saliva combinations on the in vitro growth of oral pathogenic and commensal microorganisms.

Neonates are exposed to microbes in utero and at birth, thereby establishing their microbiota (healthy microbial colonisers). Previously, we reported significant differences in the neonatal oral microbiota of breast-fed and formula-fed babies after first discovering a primal metabolic mechanism that occurs when breastmilk (containing the enzyme xanthine oxidase) and neonatal saliva (containing highly elevated concentrations of the substrates for xanthine oxidase: xanthine and hypoxanthine). The interaction of neonatal saliva and breast milk releases antibacterial compounds including hydrogen peroxide, and regulates the growth of bacteria. Using a novel in vitro experimental approach, the current study compared the effects of this unique metabolic pathway on a range of bacterial species and determined the period of time that microbial growth was affected. We demonstrated that microbial growth was inhibited predominately, immediately and for up to 24 hr following breastmilk and saliva mixing; however, some microorganisms were able to recover and continue to grow following exposure to these micromolar amounts of hydrogen peroxide. Interestingly, growth inhibition was independent of whether the organisms possessed a catalase enzyme. This study further confirms that this is one mechanism that contributes to the significant differences in the neonatal oral microbiota of breast-fed and formula-fed babies.

Androgen receptor genotyping in a large Australasian cohort with androgen insensitivity syndrome; identification of four novel mutations.

We genotyped the androgen receptor (AR) gene in 31 Australasian patients with androgen insensitivity syndrome (AIS). The entire coding region of AR was examined including analysis of polymorphic CAG and GGN repeats in all patients. AR defects were found in 66.7% (6/9) of patients with complete AIS (CAIS) and 13.6% (3/22) of patients with partial AIS (PAIS). A novel deletion (N858delG) leading to a premature stop codon was found in CAIS patient P1. CAIS patient P2 has a novel deletion (N2676delGAGT) resulting in a stop at codon 787. These mutations would result in inactivation of AR protein. A novel insertion of a cysteine residue in the first zinc finger of the AR DNA-binding domain (N2045_2047dupCTG) was found in CAIS patient P3. PAIS patient P4 has a novel amino acid substitution (Arg760Ser) in the AR ligand binding domain, which may impair ligand binding. Five patients were found to have previously reported AR mutations and no mutations were identified in the remaining patients.

Deep sequencing of the 16S ribosomal RNA of the neonatal oral microbiome: a comparison of breast-fed and formula-fed infants.

In utero and upon delivery, neonates are exposed to a wide array of microorganisms from various sources, including maternal bacteria. Prior studies have proposed that the mode of feeding shapes the gut microbiota and, subsequently the child's health. However, the effect of the mode of feeding and its influence on the development of the neonatal oral microbiota in early infancy has not yet been reported. The aim of this study was to compare the oral microbiota of healthy infants that were exclusively breast-fed or formula-fed using 16S-rRNA gene sequencing. We demonstrated that the oral bacterial communities were dominated by the phylum Firmicutes, in both groups. There was a higher prevalence of the phylum Bacteroidetes in the mouths of formula-fed infants than in breast-fed infants (p = 0.01), but in contrast Actinobacteria were more prevalent in breast-fed babies; Proteobacteria was more prevalent in saliva of breast-fed babies than in formula-fed neonates (p = 0.04). We also found evidence suggesting that the oral microbiota composition changed over time, particularly Streptococcus species, which had an increasing trend between 4-8 weeks in both groups. This study findings confirmed that the mode of feeding influences the development of oral microbiota, and this may have implications for long-term human health.

A radioimmunoassay of human prealbumin in body fluids.

Prealbumin (PA) was purified 35-fold from human serum and antibodies raised against it in rabbits. A 2-hour radioimmunoassay (RIA) using polyethyleneglycol (PEG) to separate bound and free PA was used to determine levels in body fluids. Using patient serum specimens the new method was compared with an electroimmunoassay (EIA) method and the regression equation obtained was: y = 1.13x - 9.91. The RIA and EIA methods compared favourably with respect to precision to practicability and economy. The RIA method seems especially suitable for large scale assays of PA and is 100 times more sensitive than EIA. Preliminary estimations of PA with the RIA method in plasma, cerebrospinal fluids, amniotic fluids, duodenal juices and urines were carried out. The results indicate that this method can be conveniently used to assay PA in body fluids where the protein is present in low concentration.

Quantitative determination of serum methemalbumin.

We have devised a simple, sensitive quantitative method for the determination of serum methemalbumin. The method uses a modified Allen correction to correct the alpha band of methemalbumin at 623 nm for background turbidity. The technique is robust and is more sensitive than Schumm's test.

Comparison of 51Cr EDTA clearance with formulae in the measurement of glomerular filtration rate.

The need exists for a fast and accurate method of estimating glomerular filtration rate (GFR). A number of nomograms and formulae which estimate GFR on the basis of a plasma creatinine level have appeared in the literature. We examined some of these nomograms and formulae and showed that when the formula published by Cockcroft and Gault was compared with the GFR as estimated by 51Cr EDTA clearance, the coefficient of correlation was better than 0.94. We also compared creatinine clearance with the Cockcroft and Gault formula, and the Siersbaek-Nielsen nomogram. In each case, the correlation coefficient was less than that for the Cockcroft and Gault-51Cr EDTA comparison. Because of the inherent difficulties in making accurate 24 h urine collections, and problems associated with 51Cr EDTA studies, it is suggested that the formula prediction of GFR is more reliable than a GFR estimate using a 24 h collection.

Corticotropin deficiency: a rare cause of hyponatremia mimicking SIADH.

We describe 2 patients presenting with severe chronic hyponatremia in whom clinical and biochemical features strongly suggested the syndrome of inappropriate antidiuretic hormone secretion (SIADH). Both, however, were proven to have a primary pituitary deficiency of corticotropin. Their short synacthen tests were only mildly abnormal but associated with low basal ACTH levels. The diagnosis of ACTH deficiency was made more convincingly by their dramatic response to glucocorticoid replacement therapy. In patients in whom no cause for SIADH can be found, a trial of maintenance cortisol therapy is warranted to exclude this eminently treatable condition.

A report of accidental ethylene glycol ingestion in 2 siblings.

We describe a case of 2 siblings aged 2 1/2 and 3 1/2 yrs accidentally poisoned by ethylene glycol ingestion. We found estimating the level of ethylene glycol in plasma by calculation of osmolar gap too insensitive to be of value and advocate the availability of a specific method. In our study only one of the 2 children had a toxic level of ethylene glycol but assay by conventional assay and by proton magnetic resonance spectroscopy (1HMRS) of toxic metabolites viz glycolate, glyoxylate and oxalate showed both to be excreting grossly elevated levels. This indicates the desirability of assaying the toxic metabolites of the glycol as well as the parent compound in assessing ingestions.

The variability and dietary dependence of urinary oxalate excretion in recurrent calcium stone formers.

Twenty-two recurrent calcium stone formers had 24-h urinary oxalate excretions on their home diets which were significantly greater than those of 30 normal subjects (0.48 +/- 0.23 mmol/d; mean +/- SD compared with 0.31 +/- 0.11; P less than 0.01). The stone formers also demonstrated marked day to day variability in oxalate excretion indicating that a single normal urinary oxalate measurement did not exclude significant hyperoxaluria at other times. On a hospital diet containing 1000 mg calcium per day, urinary oxalate excretion fell significantly from 0.48 +/- 0.23 mmol/d to 0.32 +/- 0.12; P less than 0.01. As the urinary calcium excretion in and out of hospital was similar, it seems unlikely that low calcium intake at home was responsible for the hyperoxaluria. All patients had recurrent symptomatic stone disease and had been advised to avoid foods rich in oxalate. Whilst poor compliance is a possible explanation for the variability in oxalate excretion, we believe it is more likely that there is an inadvertent intake of oxalogenic precursors in their diet. As normal subjects do not demonstrate hyperoxaluria on similar home diets, stone formers may have a metabolic defect in the handling of these precursors.

Equivalent metabolic acidosis with four colloids and saline on ex vivo haemodilution.

Colloid infusions can cause metabolic acidosis. Mechanisms and relative severity with different colloids are incompletely understood. We compared haemodilution acid-base effects of 4% albumin, 3.5% polygeline, 4% succinylated gelatin (all weak acid colloids, strong ion difference 12 mEq/l, 17.6 mEq/l and 34 mEq/l respectively), 6% hetastarch (non-weak acid colloid, strong ion difference zero) and 0.9% saline (crystalloid, strong ion difference zero). Gelatin weak acid properties were tracked via the strong ion gap. Four-step ex vivo dilutions of pre-oxygenated human venous blood were performed to a final [Hb] near 50% baseline. With each fluid, base excess fell to approximately -13 mEq/l. Base excess/[Hb] relationships across dilution were linear and direct (R2 > or = 0.96), slopes and intercepts closely resembling saline. Baseline strong ion gap was -0.3 (2.1) mEq/l. Post-dilution increases occurred in three groups: small with saline, hetastarch and albumin (to 3.5 (02) mEq/l, 4.3 (0.3) mEq/l, 3.3 (1.4) mEq/l respectively), intermediate with polygeline (to 12.2 (0.9) mEq/l) and greatest with succinylated gelatin (to 20.8 (1.4) mEq/l). We conclude that, despite colloid weak acid activity ranging from zero (hydroxyethyl starch) to greater than that of albumin with both gelatin preparations, ex vivo dilution causes a metabolic acidosis of identical severity to saline in each case. This uniformity reflects modifications to the albumin and gelatin saline vehicles, in part aimed at pH correction. By proportionally increasing the strong ion difference, these modifications counter deviations from pure saline effects caused by colloid weak acid activity. Extrapolation in vivo requires further investigation.

Stability of the strong ion gap versus the anion gap over extremes of PCO2 and pH.

The strong ion gap (SIG) is under evaluation as a scanning tool for unmeasured ions. SIG is calculated by subtracting [buffer base], which is ([A-]+[HCO3-), from the apparent strong ion difference, which is ([Na+]+[K+]+[Ca++]+[Mg++]-[Cl-]-[L-lactate]). A- is the negative charge on albumin and phosphate. We compared the pH stability of the SIG with that of the anion gap (AG). In normal and hypoalbuminaemic hyperlactaemic blood, PCO2 was reduced stepwise in vitro from >200 mmHg to <20 mmHg, with serial blood gas and electrolyte analyses, and [albumin] and [phosphate] measurement on completion. Respective [haemoglobin], [albumin], [phosphate] and [lactate] in normal blood were 156 (0.9) g/l, 44 (2) g/l, 1.14 (0.06) mmol/l and 1.7 (0.8) mEq/l, and in hypoalbuminaemic blood 116 (0.9) g/l, 24 (2) g/l, 0.78 (0.06) mmol/l and 8.5 (0.5) mEq/l. pH increased from < 6.85 to > 7.55, causing significant falls in [Na+] and elevations in [Cl-]. Initial and final SIG values did not differ, showing no correlation with pH. Mean SIG was 0.5 +/- 1.5 mEq/l. AG values were directly correlated with pH (normal: R2 = 0.51, hypoalbuminaemic: R2 = 0.65). Final AG values significantly exceeded initial values (normal blood: 15.9 (1.7) mEq/l versus 8.9 (1.8) mEq/l, P < 0.01; hypoalbuminaemic blood: 16.5 (0.8) mEq/l versus 11.8 (2.0) mEq/l, P < 0.05). We conclude that, unlike the AG, the SIG is not affected by severe respiratory acidosis and alkalosis, enhancing its utility in acid-base disturbances.

A comparative study of a premature infant formula and preterm breast milk for low birthweight infants.

Although the unique composition of preterm milk (PTM) has led to its increasing use in feeding of low birthweight (LBW) infants, controversy exists as to whether such milk adequately meets their requirements. This study compares the clinical tolerance and anthropometric, biochemical and haematological parameters of LBW infants fed exclusively with their own mother's PTM, a premature infant formula (Alprem; Nestlé Australia) and a mixture of PTM and Alprem. Of 90 enrolled LBW infants (1000-1750 g birthweight), 78 completed the feeding trial for a mean duration of 42 days. Twenty-eight babies were fed Alprem (Group A), 31 received a mixture of Alprem and PTM (Group B) and 18 received PTM (Group C). Babies in Groups A and B were smaller, less mature and more asphyxiated at birth than those in Group C. Weight gain from full enteral feeding was greater in Group A (18.1 g/kg per day) and Group B (17.6 g/kg per day) than in Group C (13.0 g/kg per day). Throughout the trial, weight gain in Groups A and B exceeded predicted intra-uterine growth rates, whereas that for Group C approximated the predicted intra-uterine growth rates. Growth rates of length and head circumference were also greatest in the Alprem-fed babies. Infants receiving PTM were supplemented with calcium, sodium, vitamins and energy, whereas the only three infants requiring mineral supplementation in the Alprem group were those receiving Frusemide therapy for chronic lung disease. lower serum concentrations of phosphorus, iron, albumin and urea, and higher zinc and alkaline phosphatase concentrations were found in infants receiving PTM (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Stability of ascorbate in urine: relevance to analyses for ascorbate and oxalate.

Ascorbate is unstable in urine at room temperature at pH values ranging from 1 to 12. At pH 7 and above, oxalate is generated in amounts directly proportional to the ascorbate concentration. In 12 different urines, adjusted to pH 12 and incubated for 20 h at room temperature, there was a significant correlation between the amount of oxalate formed and the initial ascorbate concentration (r = 0.97, p less than 0.01). The mean (+/- SD) concentration of oxalate (1.32 +/- 0.70 mmol/L) formed during this period approximated the initial ascorbate concentration (1.57 +/- 1.09 mmol/L). Disodium EDTA, 10 mmol/L final concentration, stabilizes ascorbate in urine and inhibits its conversion to oxalate at pH values of 4.4 to 7.0 during a 24-h period. We therefore propose that urine specimens for ascorbate and oxalate analyses be collected with disodium EDTA present such as to give about this final concentration.

Study of alanine aminopeptidase excretion as a test of gentamicin nephrotoxicity.

We studied the urinary excretion of the proximal tubular enzyme alanine aminopeptidase (AAP) by 38 patients who received gentamicin and by 45 similar control patients. AAP excretion by the control patients was highly variable. The pattern of AAP excretion distinguished patients with pre-renal and renal causes for increases in serum creatinine (SCr) but this can be done more simply with the urine to serum creatinine ratio. Peak AAP excretion did not reflect renal damage in oliguric patients and di not identify gentamicin patients with a renal cause for increases in SCr. The level of AAP excretion in the first three days of gentamicin treatment did identify the three patients with renal causes for an increase in SCr and was highest in a patient with symptomatic hypomagnesaemia due to impairment of proximal tubular function. We concluded that study of AAP excretion in normal volunteers receiving three-day courses of aminoglycosides is a valid screening test for nephrotoxicity but that AAP excretion is too non-specific to use in clinical practice.

A possible etiological role for ascorbate in calculi formation.

Studies of recurrent stone formers indicated that they have significantly increased urinary oxalate and decreased ascorbate excretions. Results of oral and intravenous administration of ascorbate indicate an enhanced production of oxalate from ascorbate in recurrent calcium stone formers as compared with normal persons and that most of this oxalate is generated in the gut.

Reference intervals for calcium, phosphate, and alkaline phosphatase as derived on the basis of multichannel-analyzer profiles.

In an attempt to demonstrate a rapid and economical approach to deriving reference intervals, we analyzed data on plasma calcium and phosphate from a multichannel analyzer for more than 20 000 subjects and data on alkaline phosphatase from more than 10 000 subjects. Subjects were selected by the criterion, that their results for constituents other than the one of interest were within current reference intervals. Thus we have been able to include older patients who have diseases that accompany old age, but that do not affect test results. The mean concentrations of calcium and phosphate decreased with increasing age in both sexes, except for an abrupt increase for women about the time of reaching menopause. Similarly, the mean alkaline phosphatase activity increased with age in both sexes, reflecting a skewed frequency distribution. Here also, there was an abrupt increase in the modal value for women near menopause.

Evidence that the oral glucose-tolerance test does not provide a uniform stimulus to pancreatic islets in pregnancy.

Fifty consecutive pregnant patients referred for a glucose-tolerance test were classified on the basis of increasing (n = 20) or decreasing (n = 28) hematocrit after an oral 75-g glucose load. (The hematocrit did not change in the other two patients.) Patients with increasing hematocrit, a response previously seen in patients with the dumping syndrome, showed significantly flatter increases in glucose concentrations in plasma after the load. The mean decrease in the concentration of phosphate in plasma, measured as an index of glucose uptake by cells, was significantly less (P less than 0.05) 2 h after the load in the group with flatter glucose responses, suggesting that the flat response is ascribable to poor glucose absorption rather than to an exaggerated insulin response. These results indicate that the oral glucose-tolerance test stresses the pancreatic islets differently in different pregnant subjects, owing to individual variations in the gastrointestinal handling of the glucose load. Consequently, patients may give a "normal" result who might otherwise become hyperglycemic after normal meals. We suggest that alternative screening procedures be investigated to assess pregnant patients postprandially.