CYP21 and CYP21P variability in steroid 21-hydroxylase deficiency patients and in the general population in the Netherlands.

Steroid 21-hydroxylase deficiency is caused by defectiveness of the CYP21 gene. Such defects have presumably originated from interactions with the nearby CYP21P pseudogene during evolution. We studied these mechanisms by comparing the genetic variability of CYP21, CYP21P, and CYP21P/CYP21 hybrids (resulting from large-scale rearrangements) at eight mutation sites in a group of Dutch steroid 21-hydroxylase deficiency patients, their family members, and controls. The most common CYP21 defect in patients with salt-losing steroid 21-hydroxylase deficiency was a splice junction mutation in intron 2. The most common defect in the simple virilising form of the disease was ile72 --> asn. CYP21P showed considerable sequence variation in its central and 3' sections; the 5' section was constant. A single nucleotide (T) insert in exon 7 was found in all CYP21P genes. During the course of evolution, this was probably the third defect introduced into CYP21P after the splice junction mutation in intron 2 and the 8 bp deletion in exon 3. Gene conversions introducing CYP21-like sequences contribute to CYP21P variability. Such an event has occurred de novo in one family. A comparison of CYP21 and CYP21P mutations on the same chromosome shows that at least some of the small-scale gene conversions that supposedly transfer defects to CYP21 involve interaction between homologous chromosomes. The majority of the putative CYP21P-CYP21 transitions in hybrid genes appears to occur in a distinct zone that lies 5' of nucleotide 2108, which is further downstream than previously hypothesised. The other transitions lie upstream of nucleotide 999. Apparent 'large-scale' CYP21-CYP21P gene conversions lead to hybrid genes that are very similar to those found in CYP21 deletions, so these haplotypes have probably resulted from a meiotic double unequal crossover.

Energy utilization and growth in breast-fed and formula-fed infants measured prospectively during the first year of life.

This study is the first to report approximations of energy requirements for male and female breast-fed and formula-fed infants based on individual estimates of total daily energy expenditure (TDEE) and energy deposition derived from total body fat (TBF) and fat-free mass (FFM) gain as determined by total-body electrical conductivity. In 46 healthy, full-term infants the effect of > or = 4 mo of exclusive breast-feeding compared with formula feeding on macronutrient and energy intake, TDEE, energy deposition, and growth were investigated prospectively. Metabolizable energy intake (MEI) was assessed from macronutrient intake by test weighing (MEI-TW) and from the sum of TDEE and energy deposition (MEI-Pred). At 1-2, 2-4, 4-8, and 8-12 mo of age MEI-Pred averaged 431 +/- 38, 393 +/- 33, 372 +/- 33, and 355 +/- 21 kJ x kg(-1) x d(-1) for boys, and 401 +/- 59, 376 +/- 25, 334 +/- 33, and 326 +/- 17 kJ x kg(-1) x d(-1) for girls. No significant difference between breast-fed and formula-fed infants was found with respect to weight, length, head circumference, TBF, FFM, and TDEE at all ages, or for gain in length, weight, TBF, and FFM. MEI-TW was significantly different between feeding groups at 1-4 mo of age (formula-fed being greater than breast-fed, P < 0.005). This feeding effect, however, was not significant for MEI-Pred (MJ/d). MEI-TW differed from MEI-Pred only in breast-fed infants at 1-4 mo (P < 0.05 at 2-4 mo). The data from this study indicate that energy requirements in infants are lower than the recommendations in guidelines currently in use.

Aldosterone production despite absence or defectiveness of the CYP21 genes in two patients with salt-losing congenital adrenal hyperplasia caused by steroid 21-hydroxylase deficiency.

Aldosterone and cortisol were found in plasma samples from two patients with salt-losing congenital adrenal hyperplasia caused by steroid 21-hydroxylase deficiency. One patient had a CYP21 gene deletion on one chromosome and a mutation causing erroneous mRNA splicing on the other. The other patient had a CYP21 gene deletion on one chromosome and a large scale conversion of CYP21 to CYP21P on the other. All CYP21P-like genes in these patients were defective, since they carried a deleterious 8 bp deletion in the third exon. After HPLC purification of the patients' plasma samples, cortisol was no longer detectable in the radioimmunoassay, but aldosterone levels were still within or slightly above the normal reference range. Aldosterone dropped to very low levels after steroid replacement therapy had taken effect. In at least one of these patients, the genetic defect rules out normal functioning of the adrenocortical steroid 21-hydroxylase, which implies involvement of an alternative enzyme system.

Structural position and amount of palmitic acid in infant formulas: effects on fat, fatty acid, and mineral balance.

The structure of the triglycerides (TG) in human milk (HM) differs from those of vegetable oils used in infant formulas. In HM, palmitic acid is predominantly esterified to the center or beta-position of the TG, in vegetable oil, it is mainly at the external or alpha-positions. These differences in configuration affect intestinal fat absorption. Fat and mineral balances were investigated in three groups of 9 healthy term infants aged 5 weeks. Infants were randomly assigned to receive one of the three study formulas from birth: (a) formula beta, resembling the structure of HM fat most closely (24% palmitic acid, 66% esterified to beta-position), (b) formula intermediate (24% palmitic acid, 39% esterified to the beta-position), and (c) regular formula (20% palmitic acid; 13% esterified to the beta-position). Fat absorption was highest in infants fed the beta formula (97.6 +/- 0.9%), intermediate in those fed with the intermediate formula (93.0 +/- 1.8%), and lowest in infants receiving the regular formula (90.4 +/- 4.6%). Fecal calcium excretion was significantly lower in the beta group than in the other two groups (43.3 +/- 18.1 vs. 59.9 +/- 15.1 vs. 68.4 +/- 22.3 mg.kg-1.day-1 for beta, intermediate, and regular respectively). Dietary TG containing palmitic acid predominantly at the beta-position, as in HM, have significant beneficial effects on the intestinal absorption of fat and calcium in healthy term infants.

The very low birth weight premature infant is capable of synthesizing arachidonic and docosahexaenoic acids from linoleic and linolenic acids.

Infants fed formulas devoid of long-chain polyunsaturated fatty acids (LCP) exhibit low plasma LCP concentrations and have poorer retinal and neurologic development in comparison with their human milk-fed counterparts. It is not known whether the low plasma LCP concentrations result from an impaired biosynthetic capacity, a high need or a low dietary intake. With stable isotope technology and high sensitivity tracer detection using gas chromatography-isotope ratio mass spectrometry we measured the conversion of [13C]linoleic acid (C18:2n-6) and [13C]linolenic acid (C18:3n-3) into their longer chain derivatives in five 1-mo-old formula-fed preterm infants (birth weight 1.17 +/- 0.12.kg and gestational age 28.4 +/- 1.3 wk). Carbon-13-labeled linoleic acid and inolenic were mixed with the formula and administered continuously for 48 h. Both tracers were rapidly incorporated in plasma phospholipids, and their metabolic products including arachidonic acid (C20:4n-6) and docosahexaenoic acid (C22:6n-3) became highly enriched. We demonstrate that the preterm infant is capable of synthesizing LCP from their 18-carbon precursors, and our data do not support the hypothesis that a reduced delta 6 desaturation is a main factor leading to low arachidonic acid and docosahexaenoic acid levels.

Standards for total body fat and fat-free mass in infants.

Data on body composition in conjunction with reference centiles are helpful in identifying the severity of growth and nutritional disorders in infancy and for evaluating the adequacy of treatment given during this important period of rapid growth. Total body fat (TBF) and fat-free mass (FFM) were estimated from total body electrical conductivity (TBEC) measurements in 423 healthy term Caucasian infants, aged 14-379 days. Cross sectional age, weight, and length related centile standards are presented for TBF and FFM. Centiles were calculated using Altman's method, based on polynomial regression and modelling of the residual variation. The TBF percentage steeply increased during the first half year of life, and slowly declined beyond this age. Various simple TBEC derived anthropometric prediction equations for TBF and FFM are available to be used in conjunction with these standards. Regression equations for the P50 and the residual SD, depending on age, weight, or length, are provided for constructing centile charts and calculating standard deviation scores.

Density of eosinophils reflects activity of disease in allergic asthmatic children.

BACKGROUND: Low density eosinophils are more prominent in asthmatic patients compared with healthy subjects. LDE are metabolically more active and produce more tissue-injuring and spasmogenic proteins than normal eosinophils. OBJECTIVE AND METHODS: With a method providing information about eosinophils of 12 different densities we were able to study eosinophil density characteristics in 24 young patients in detail with allergic asthma in a stable phase, and in 21 patients after a bronchial allergen challenge. RESULTS: Study of the eosinophil density profile of patients and healthy controls revealed two density populations. Patients had more low density eosinophils than controls. In the patients eosinophil density characteristics and in particular the number of low density eosinophils correlated strongly with both FEV1% predicted (p = -0.66, P < 0.001) and FEV1/FVC (p = -0.47, P < 0.01) as well as with bronchial responsiveness to histamine (p = -0.68, P < 0.001) and house dust mite (p = -0.37, P < 0.05). Allergen induced bronchial reactions were associated with an increase in the number (P < 0.001) and percentage (P < 0.05) of low density eosinophils. A selective rise in the number of eosinophils collected from fractions with a low density accounted for the observed rise in the total number of eosinophils. Density changes did not differ between patients with an isolated early reaction and patients with both an early and a late reaction, nor was there a relation between the severity of the late reaction and the shift in eosinophil density. CONCLUSION: In conclusion, peripheral blood eosinophil density characteristics and in particular numbers of low density eosinophils are closely related with indicators of the asthma severity under stable conditions. Allergen inhalation induces a further shift towards lower density suggesting additional activation of the eosinophils.

Effect of dietary triacylglycerol fatty acid positional distribution on plasma lipid classes and their fatty acid composition in preterm infants.

Seven premature infants were each fed, for 1 wk in a crossover design. The beta formula contained triacylglycerols resembling the stereoisomeric structure of human milk fat (25.4% by wt 16:0, 76.1% of which is at the sn-2 position), whereas in the alpha formula 87.3% of total 16:0 (25.7% by wt was at the sn-1,3 positions. Plasma lipids and their fatty acid compositions were determined at the end of each 1 wk study period. Infants fed with the beta formula had higher percentages of palmitic acid in plasma sterol esters, triacylglycerols, and free fatty acids, and lower linoleic acid in triacylglycerols than with the alpha formula. Premature infants fed formulas with triacylglycerols 16:0 predominantly in the sn-2 rather than the sn-1,3 positions had alterations in their plasma fatty acids consistent with enhanced absorption of 16:0 from the sn-2 compared with the sn-1,3 positions.

Measurement of fat-free mass in infants.

Body composition data are important for adequate monitoring of growth and nutritional status in infants. 18O Isotope dilution techniques (ID18-O) are widely used to estimate total body water (TBW) and calculate fat-free mass (FFM). A problem of isotope dilution is an underestimation of TBW by the extrapolation to t = O approach and an overestimation of TBW by the plateau approach. Using total body electrical conductivity (TOBEC) as the reference technique we validated the extrapolation approach by 149 measurements (boys, n = 76; girls, n = 73) in 50 healthy infants aged 1-12 mo. TOBEC-derived FFM and fat mass were in excellent agreement with Fomon's reference data. Strictly linear relationships with slopes not significantly different from one were found between FFM estimated by TOBEC (FFMTOBEC) and FFM estimated by ID18-O (FFM18-O) (r = 0.98 and residual SD = 0.29 for boys, r = 0.98 and residual SD = 0.32 for girls). FFM18-O was slightly but significantly lower than FFMTOBEC, the difference being on average 0.18 (+/- 0.24) kg for girls and 0.08 (+/- 0.21) kg for boys (i.e. respectively 4 (+/- 4.5)% (p < 0.0001) and 1.5 (+/- 3.9)% (p = 0.004) of FFMTOBEC). We conclude that ID18-O using the extrapolation to t = O approach is suitable for TBW and FFM estimations in groups of infants. Due to the considerable measurement error of ID18-O (estimated at approximately 6%), individual TBW18-O and FFM18-O estimates should be considered with some caution.

Body fat and fat-free mass in infants: new and classic anthropometric indexes and prediction equations compared with total-body electrical conductivity.

Anthropometry is frequently used for nutritional assessment. Little is known in infants about the validity of anthropometric measurements in relation to whole-body fat (TBF) and fat-free mass (FFM) composition. We compared TBF and FFM estimations by total-body electrical conductivity (TOBEC) with anthropometry in 435 healthy infants ages 21-365 d. TBF was best correlated with weight-for-length and calf circumference (r2 = 0.84, r2 = 0.83). FFM was best correlated with body weight (r2 = 0.93). Upper-arm anthropometry, skinfold thickness, and Quetelet's and Ponderal indexes were poorly correlated with TBF and FFM (r2 < 0.65). New anthropometry-based prediction equations were calculated (r2 = 0.90 for TBF and r2 = 0.95 for FFM). New simple indexes (analogous to Quetelet's index) were calculated for TBF (weight x calf circumference/length; r2 = 0.87) and for FFM (square root of weight x length; r2 = 0.95). Prediction equations and indexes were cross-validated in a second population by a second observer. Interobserver variation was largest for equations with skinfold thicknesses included. We conclude that anthropometry can be used for rough estimations of infant body composition, although indexes different than those used in children and adults are preferred.

Feeding premature newborn infants palmitic acid in amounts and stereoisomeric position similar to that of human milk: effects on fat and mineral balance.

The effect of the structure of human milk triglycerides on intestinal fat absorption remains controversial. Twelve infants were each fed, for 1 wk in a crossover design, two formulas that differed only in triglyceride configuration. The "beta" formula contained triglycerides similar to those in human milk (26% palmitic acid, esterified predominantly to the sn-2 position) whereas in the "alpha" formula, which contained triglycerides similar to those in formulas currently marketed, palmitate was mainly at the sn-1,3 positions. Fatty acid, fat, and mineral balances were measured at the end of each 1-wk period. Myristic, palmitic, and stearic acids were absorbed better from the beta formula, but total fat excretion was not reduced. During the feeding of beta formula fecal calcium excretion was lower, urinary calcium higher, and urinary phosphate lower. A formula containing triglycerides similar to those in human milk has significant effects on fatty acid intestinal absorption and improves mineral balance in comparison with a conventional formula.

Whole-body protein turnover in preterm appropriate for gestational age and small for gestational age infants: comparison of [15N]glycine and [1-(13)C]leucine administered simultaneously.

Measurements of whole-body protein turnover in preterm infants have been made using different stable isotope methods. Large variation in results has been found, which could be due to different clinical conditions and/or the use of different tracers. We studied 14 appropriate for gestational age and nine small for gestational age orally fed preterm infants using [15N]glycine and [1-(13)C]leucine simultaneously, which allowed us to make a comparison of commonly used methods to calculate whole-body protein turnover. Whole-body protein turnover was calculated from 15N enrichment in urinary ammonia and urea after [15N]-glycine administration and from the 13C enrichment in expired CO2 after administration of [1-(13)C]leucine. Enrichment of alpha-ketoisocaproic acid after [1-(13)C]leucine constant infusion was measured as a direct parameter of whole-body protein turnover. Group means for whole-body protein turnover using [15N]glycine or [1-(13)C]leucine ranged from 10 to 14 g.kg-1.d-1, except when using the end product method that assumes a correlation between leucine oxidation and total nitrogen excretion. We found very low 15N enrichment of urinary urea in the majority of small for gestational age infants. These infants also had a lower nitrogen excretion in urine and oxidized less leucine. Nitrogen balance was higher in small for gestational age infants (416 +/- 25 mg.kg-1.d-1) compared with appropriate for gestational age infants (374 +/- 41 mg.kg-1.d-1, p = 0.003). [15N]Glycine does not seem to exchange its label with the body nitrogen pool to a significant degree and is therefore not always suitable as a carrier for 15N in protein turnover studies in premature infants.

Quantitative assessment of infant body fat by anthropometry and total-body electrical conductivity.

Measurement of total-body electrical conductivity (TOBEC) has emerged as a rapid, safe, and reproducible method for estimation of infant total body fat (TBF). Agreement of two anthropometric methods [by Dauncey et al (1977) and Weststrate et al (1989)] with TOBEC-TBF was assessed in 435 healthy infants aged 21-365 d. Dauncey-TBF correlated with TOBEC-TBF by r2 = 0.61 and exceeded TOBEC-TBF by 0.14 +/- 0.25 kg in infants < 4 mo of age. Thereafter, TOBEC-TBF exceeded Dauncey-TBF by 0.20 +/- 0.47 kg. We modified Dauncey's method, which significantly improved the correlation to r2 = 0.75. Weststrate-TBF correlated with TOBEC-TBF by r2 = 0.87, but exceeded TOBEC-TBF by 0.5 kg. Both methods showed poor agreement with TOBEC-TBF. We conclude that both methods, although suitable for comparison of TBF between groups, cannot be used to accurately assess TBF in an individual infant.

Total body electrical conductivity measurements: an evaluation of current instrumentation for infants.

Quantitation of the body's fat and learn masses is an important component of nutritional assessment. Such measurements, however, are difficult to conduct routinely in infants due to the numerous limitations of traditional methods. The application of total body electrical conductivity measurements for quantitating fat-free mass (FFM) overcomes many of these limitations. The instruments required to perform these measurements in pediatric patients (HP-2) have recently become commercially available, but their measurement performance has not been evaluated. In these studies, we compared the precision, day-to-day variability, and magnetic field profile of three HP-2 instruments. We also derived a new calibration equation that relates the FFM to the total body electrical conductivity measurement in piglets, and compared it with an equation (provided currently by the manufacturer) derived on a prototype instrument. The performance of the instruments was generally similar, although a significant difference in the magnetic field of one instrument was identified. The coefficient of variation of inanimate phantom measurements varied from +/- 0.2 to +/- 0.5%, and the day-to-day variability was generally similar. Such measurement error is significant (+/- 0.035 to +/- 0.078 kg FFM) for small subjects. The new calibration equation was similar to the original equation; therefore, all the data were pooled to generate a new equation that is linear at least to 10 kg. Thus, the HP-2 total body electrical conductivity instruments, which can be safely and easily used to measure FFM and fat in infants through 1 y of age, proved to be reliable and precise, and results obtained from different instruments can be confidently compared.

Conversion of octanoic acid into long-chain saturated fatty acids in premature infants fed a formula containing medium-chain triglycerides.

A large number of very-low-birth weight infants are fed formulas containing medium-chain triglycerides (MCT) to enhance fat and calcium absorption. Studies are available on the intestinal absorption of MCT, which is nearly complete, but uncertainties exist on the metabolic fate of octanoic acid, the major component of MCT. Oxidation accounts for approximately 50% of the dietary intake, and losses as dicarboxylic acids in the urine are negligible. Since storage in adipose tissue is limited, conversion into long-chain fatty acids (LCFA) is likely to be an important route. To study the nonoxidative metabolism of MCT, six preterm infants fed a standard premature formula containing 38 weight% (wt%) MCT (54 mol% medium-chain fatty acids (MCFA), of which 35 mol% is octanoic acid) were studied at 4 weeks of age, when on full oral intake and receiving on average 130 kcal/kg/d. The study consisted of an oral primed constant-rate infusion of [13C]-octanoate and the measurement of the 13C enrichment of individual fatty acids in plasma triglycerides (TG) by a highly sensitive on-line combustion method using gas chromatography-isotope ratio mass spectrometry (GC-IRMS). We observed a significant incorporation of the dietary [13C]-octanoic acid in plasma TG (10.0% +/- 4.5% of the enrichment of the diet). A noticeable incorporation of the label was detected in myristic and palmitic acids (4.6% +/- 2.5% and 7.8% +/- 4.1% of the octanoic enrichment of the diet). The absolute amount of the fatty acids was studied with conventional GC, and the plasma TG fatty acid profile differed markedly from the diet.(ABSTRACT TRUNCATED AT 250 WORDS)

A practical approach to the detection of androgen receptor gene mutations and pedigree analysis in families with x-linked androgen insensitivity.

Androgen insensitivity syndrome (AIS) is an X-linked disorder in which defects in the androgen receptor gene have prevented the normal development of both internal and external male structures in 46,XY individuals. This survey reports the analysis of 11 AIS subjects. The androgen receptor gene of these subjects was analyzed using polymerase chain reaction (PCR)-single-strand conformation polymorphism analysis and sequencing or sequencing of PCR-amplified androgen receptor gene fragments alone. In total, 10 single base changes and one partial gene deletion were detected. Seven single base changes resulted in an amino acid change, one resulted in the introduction of a premature stop codon, one event represented a single base insertion resulting in a frame-shift, and one single base change affected a donor splice site. The androgen receptor protein in genital skin fibroblasts from several patients was studied with respect to molecular mass after immunoprecipitation and SDS-PAGE. Two patients expressed a truncated receptor protein in agreement with the established genomic mutation. Pedigree analysis was performed to identify possible carriers for the syndrome in families of AIS patients using single-strand conformation polymorphism and restriction site analysis of PCR products. In one case, the polymorphic (CAG)n(CAA) repeat in exon 1 encoding a polyglutamine stretch was used to identify the mutant allele in a family with X-linked partial androgen insensitivity before the identification of the actual genomic mutation. PCR-single-strand conformation polymorphism analysis proved to be a fast and reliable technique to screen for androgen receptor gene mutations and to study the androgen receptor gene of family members of AIS-affected individuals.

The effect of alterations in physical and chemical characteristics on TOBEC-derived body composition estimates: validation with non-human models.

The measurement of total-body electrical conductivity (TOBEC) has become one of the standard methods for the estimation of body composition in infants. We investigated, using non-human models, the effect on the accuracy of TOBEC-derived body composition estimates of alterations in physical and chemical characteristics of the fat-free mass (FFM). The effect of electrolyte type, concentration and volume on TOBEC was determined using 2, 3 and 51 solutions of six different chlorides and sodium bicarbonate. Equimolar concentrations yielded TOBEC values in accordance with known ion conductivities: H+ >> Ca2+ > Mg2+ > K+ > Na+ > Li+ and Cl- > HCO3-. The behaviour of these solutions was described very accurately over a wide range of concentrations (1-200 mM) by a simple exponential law. Dissolved egg-white protein, glycine and L-glutamine elicited no TOBEC signal. In vitro, using polyethylene bottles filled with physiologic saline, in the interval of 2-45 degrees C a linear relation was observed between temperature and TOBEC. Below the freezing point no TOBEC signal was elicited. The effect of tissue autolysis and body temperature on TOBEC was examined by repeated measurements of TOBEC and temperature in seven fresh infant minipig cadavers. Five minipigs were allowed to cool. Shortly after death TOBEC decreased by 2.5% per degrees C. Two animals were kept at constant temperature. The TOBEC signal showed a gradual increase of 9% after 7 h due to autolysis. We conclude that in vivo TOBEC measurements are affected by ion concentration (e.g. non-isotonic hydration changes), geometry (e.g. deviations in body shape), temperature (e.g. fever, skin cooling) and tissue autolysis (measurements after death). Proteins, molecules with strong dipole moments and ions trapped in crystalline structures do not significantly affect the TOBEC reading.

Differential splicing of human androgen receptor pre-mRNA in X-linked Reifenstein syndrome, because of a deletion involving a putative branch site.

The analysis of the androgen receptor (AR) gene, mRNA, and protein in a subject with X-linked Reifenstein syndrome (partial androgen insensitivity) is reported. The presence of two mature AR transcripts in genital skin fibroblasts of the patient is established, and, by reverse transcriptase-PCR and RNase transcription analysis, the wild-type transcript and a transcript in which exon 3 sequences are absent without disruption of the translational reading frame are identified. Sequencing and hybridization analysis show a deletion of > 6 kb in intron 2 of the human AR gene, starting 18 bp upstream of exon 3. The deletion includes the putative branch-point sequence (BPS) but not the acceptor splice site on the intron 2/exon 3 boundary. The deletion of the putative intron 2 BPS results in 90% inhibition of wild-type splicing. The mutant transcript encodes an AR protein lacking the second zinc finger of the DNA-binding domain. Western/immunoblotting analysis is used to show that the mutant AR protein is expressed in genital skin fibroblasts of the patient. The residual 10% wild-type transcript can be the result of the use of a cryptic BPS located 63 bp upstream of the intron 2/exon 3 boundary of the mutant AR gene. The mutated AR protein has no transcription-activating potential and does not influence the transactivating properties of the wild-type AR, as tested in cotransfection studies. It is concluded that the partial androgen-insensitivity syndrome of this patient is the consequence of the limited amount of wild-type AR protein expressed in androgen target cells, resulting from the deletion of the intron 2 putative BPS.