Determination of glucitollysine for the quantitation of non-enzymatic glucosylation by ion exchange chromatography and reverse phase liquid chromatography.

A specific and sensitive method for the quantitative determination of the stable, reduced glucose-lysine adduct, glucitollysine (GL), in plasma protein samples is described. The method uses standard amino acid ion exchange chromatography followed by reverse phase high performance liquid chromatography after derivatisation of GL to a fluorescent product. Moreover, GL was characterised and identified in plasma samples by means of mass spectroscopy. GL measured in plasma samples of eleven type I diabetics and two healthy controls showed a significant linear correlation to concomitantly determined haemoglobin AI and glucosylated plasma proteins, but did not correlate with plasma glucose levels. This method allows the estimation of non-enzymatic glucosylation in biological samples with a high degree of specificity and sensitivity down to the low nanogram range.

Inhibition of alkaline phosphatase activity by glucose.

Non-enzymatic glycosylation (NEG) of alkaline phosphatase (AP) was studied after short- and long-term incubation with glucose and other carbohydrates. Glucose and amino sugars clearly inhibited the enzyme activity; this was in contrast to reducing and non-reducing disaccharides, which had an enhancing effect. After AP had been incubated with 18 nmol/l glucose for 180 minutes (short-term incubation), a subsequent extensive dialysis revealed full recovery of the enzymatic activity. This, plus the demonstration of a [3H]sodium borohydride-reducible glucose-protein adduct, indicated that initially a labile aldimine (Schiff base) had been formed. Binding experiments with [14C]glucose and failure of dialysis to achieve a recovery of enzymatic activity after long-term incubation suggested that subsequently a stable ketoamine product had been formed. This was further confirmed by the thiobarbituric acid test, which revealed 0.65 nmol 5-hydroxymethylfurfural/mg protein for glycosylated AP compared to 0.11 for the non-glycosylated control. Preliminary results further suggest that NEG of AP also occurs in vivo. Streptozotocin diabetic rats had significantly lower serum AP activities than did non-diabetic controls (mean +/- SD: 153.7 +/- 28.4 vs. 760.5 +/- 95.7 U/l; p less than 0.001). Blood glucose levels and serum AP activity, which had been determined simultaneously during an oral glucose tolerance test, showed without exception an inverse relationship in each of 32 healthy children studied. The biological significance of these findings remains to be established.

[Bronchopulmonary dysplasia: X-ray patterns and grading according to stages (author's transl)]. / Die bronchopulmonale Dysplasie--Röntgensymptomatik und Stadieneinteilung.

Bronchopulmonary dysplasia, first described by Northway in 1967 as a separate entity, was graded into four stages. Basing on thoracic x-ray films of 14 newborn and prematurely born infants an attempt has been made to analyse the x-ray patterns of signs characterising bronchopulmonary dysplasia and to establish a relation between the x-ray signs and the four stages. With few exceptions the various radiologic signs were seen in all stages, although with varying incidence. It follows that x-ray visualisation of the thorax yields limited pointers only to the actual stage of the disease. Roentgenography is the method of choice (in connection with the pattern of clinical signs) for diagnosing bronchopulmonary dysplasia and should be also employed for controlling the course of the disease and for the identification of eventual complications.

[Neonatal small left colon syndrome: a finding of only radiological or also clinical significance? (author's transl)]. / Neonatal Small Left Colon Syndrome: ein nur radiologischer oder auch klinisch relevanter Befund?

The finding in the literature that the neonatal small left colon syndrome can be present in newborn infants of diabetic mothers without relevant clinical signs led us to perform contrast enema investigations of the colon in 20 newborn infants of insulin-dependent diabetic mothers. Among these only one infant, with signs of intestinal obstruction, had the small left colon syndrome. During the same period the neonatal small left colon syndrome was diagnosed in 4 out of 10 mature newborn infants of healthy, non-diabetic mothers who underwent contrast enema investigation because of intestinal obstruction. The concepts of the pathogenesis of the neonatal small left colon syndrome and the importance of this form of functional intestinal obstruction in the newborn are discussed together with the presumptive relationship to severe gastrointestinal complications such as e.g. necrotizing enterocolitis, seen with increasing frequency in artificially ventilated newborns.

[Cholestatic jaundice and hypophosphataemia in parenterally-fed premature infants--coincidence or causal connection? (author's transl)]. / Cholestatischer Ikterus und Hypophosphatämie bei parenteral ernährten Frühgeborenen. Zufälliges Zusammentreffen oder ätiologisch wirksamer Mechanismus?

A report is presented of the chemical pathological findings in 14 premature and one full-term infant receiving almost exclusively parenteral nutrition during the first two weeks of life. Six infants developed cholestatic jaundice. The underlying diseases were the idiopathic respiratory distress syndrome in 10, gastroschisis in 3 and renal insufficiency in 1, while one was an otherwise healthy small for dates infant. After parenteral nutrition had been discontinued obstructive jaundice cleared by the third month of life except in one infant. Apart from the higher glucose intake during the second week, jaundiced infants principally differed from non-jaundiced infants by the development of significant hypophosphataemia. An attempt was made to correlate aetiologically the presence of cholestatic jaundice with the finding of hypophosphataemia on the possible basis of a disturbance of energy metabolism.

[Heroin withdrawal syndrome in a newborn infant (author's transl)]. / Heroin-Entzugssymptomatik bei einem Neugeborenen.

The case of a newborn infant showing heroin withdrawal syndrome is reported. Symptoms of neonatal drug withdrawal syndrome, together with the complex problem of maternal drug addiction and pregnancy are briefly discussed.

[A comparison of Ultrax, Diazil, Bactrim and Spiramycin in experimental toxoplasmosis in mice (author's transl)]. / Vergleichende Untersuchung der Wirksamkeit von Ultrax, Diazil, Baktrim und Spiramycin auf die experimentelle Toxoplasmose der Maus.

White mice infected intraperitoneally with the RH-strain of Toxoplasma gondii (inoculum size 50,000 to 100,000 free protozoans per mouse) received treatment between the second and eighth day after infection with sulphamethazine-pyrimethamine, sulphamethoxy-diazine-pyrimethamine, trimethoprim-sulphamethoxazole or spiramycin subcutaneously. All untreated controls and all mice of the trimethoprim-sulphamethoxazole and spiramycin-treated groups died during the acute stage (except two mice in the latter group on the 15th day). The mean survival times were 6.5, 7.5 and 7.6 days, respectively. The best results were obtained in the sulphamethazine-pyrimethamine-treated mice; 18 out of 27 survived the 30-day observation period (5 cured), in contrast to only 9 out of 40 sulphamethoxydiazine-pyrimethamine-treated mice. Considering the high pathogenicity of the RH-strain, the differences in immunological defence mechanisms in mice (absence of antibody-activating "accessory factor") and the late commencement of treatment of the infected mice, one can state that the combination of sulphamethoxydiazine-pyrimethamine should also be capable of overcoming acute human toxoplasmosis in pregnancy. Spiramycin, by contrast, should be given only in cases where sulphonamide intolerance exists and must then be given in high doses until delivery. The combination of sulphamethoxazole-trimethoprim cannot be recommended.

[Is congenital hour-glass bladder always a malformation? (author's transl)]. / Angeborene Sanduhrblase stets ein Bildungsfehler?

Hour-glass bladder was detected by X-ray investigation in a newborn female infant with delayed postpartum micturition. No signs of difficulty in micturition or urinary tract infection were subsequently detected. Control X-ray investigation at the age of 5 1/2 months showed a completely normal-shaped urinary bladder. This finding contrasts with the general opinion that hour-glass bladder is caused by a congenital malformation.

[Congenital toxoplasmosis in two newborn infants (author's transl)]. / Angeborene Toxoplasmose bei 2 Neugeborenen.

Two newborn infants with congenital toxoplasmosis despite serological testing during pregnancy were observed at our premature and neonatal intensive care unit within a short time of one another. In one case the counter-regulatory late first test, performed only in the 26th week, was positive with a high titre. The immediately recommended treatment was not carried out. Apart from the signs of congenital generalized infection, the newborn infant already manifested chorioretinitis and encephalitis. In the second case the initially serologically-negative pregnant women became infected only during the 35th to 36th week of gestation, around the time of the third serological examination. The child was born with slight signs of general infection, but without CNS involvement. Immediate postnatal treatment resulted in complete cure. This shows that such rare cases which can no longer be detected by serological testing can be treated postnatally with good results.

[Association between the glomerular basement membrane and fibronectin as revealed by affinity chromatography ]. / Bindung zwischen Glomerulum-Basalmembran und Fibronectin Eine affinitätschromatographische Studie.

Though there is sufficient evidence that fibronectin is an integral extracellular matrix protein of the normal human kidney, the distribution and the interaction with structural proteins of the kidney have not been resolved. There are disagreements between investigators whether fibronectin is a component of the glomerular basement membrane or linked to it. We have been examining the interaction between the glomerular basement membrane and its components type IV collagen and laminin and fibronectin. Applying affinity chromatography we detected significant interaction between collagen type IV and laminin, and fibronectin on the other hand. The noncovalent interaction between collagen type IV and fibronectin was reversible after elution with 2 M urea, the association between laminin and fibronectin was even stronger and reversible applying 3 M urea for elution. The glomerular basement membrane-fibronectin interaction reflected the laminin pattern though minor binding sites at 1 M urea and 2 M urea could be detected. These interaction studies showed the chemical and thermodynamical possibilities and probability of the glomerular basement membrane and fibronectin linkage.

[Interacting between amyloid P and connective tissue proteins ]. / Wechselwirkungen zwischen Amyloid P und Bindegewebsproteinen.

In order to look for the position of amyloid P in the macromolecular connective tissue and extracellular matrix system, we performed binding studies involving affinity chromatography. Binding studies revealed the strong binding of fibronectin to amyloid P (S-AP). The fibronectin-amyloid P linkage was dissociated after elution with 2 M urea. Heparan sulfate, a major glycosaminoglycan of the extracellular matrix, showed strong binding to S-AP, which was dissociated at 3 M urea. Laminin, collagen type I and type IV, reduced and alkylated glomerular basement membranes as well as the glycosamino-glycans hyaluronic acid and chondroitin-4-sulfate failed to bind to S-AP. Our binding studies show that amyloid P can react strongly with extra cellular matrix proteins and can help to explain the presence of amyloid P in normal connective tissue.

[Thyroid function in healthy premature infants and in premature infants with the hyaline membrane syndrome ]. / Schilddrüsenfunktion bei gesunden Frühgeborenen und bei Frühgeborenen mit hyalinem Membransyndrom.

The serum concentrations of thyrotropin (TSH), thyroxine (T4), trijodothyronine (T3) and of 3,3',5', trijodothyronine (reverse T3, rT3) were followed up during the first 4 weeks of life in 5 healthy premature infants and in 6 premature infants with hyaline membrane syndrome and artificial ventilation. TSH and T4 concentrations remained unchanged in both groups. T3 levels increased during the observation period and were significantly lower in the sick prematures on days 1 and 3 (p less than 0,02). T3 and the rT3/T3 ratio was increased in healthy and in the sick prematures, the sick prematures showing higher values, and decreased in both groups during the first week of life. We found no significant correlation between TSH and the peripheral thyroid hormone levels as well as no relation between the respiratory compliance-indicating degree of lung maturity-and the T3 or T4 concentrations. Postpartal T3 or T4 serum levels were correlated significantly with the duration of artificial ventilation. Our data indicate that a "low T3 syndrome" was present in all prematures after birth and that respiratory distress syndrome increased conversion of T4 to rT3. From our results we cannot conclude that substitution therapy with thyroid hormones may be useful in premature infants with the hyaline membrane syndrome.

[Non-enzymatic glycosylation of hemoglobin and serum protein in children with galactosemia]. / Nichtenzymatische Glykosylierung von Hämoglobin und Serumprotein bei Kindern mit Galaktosämie.

Non-enzymatic galactosylation has been investigated by in vitro incubation of red cell haemolysate, a HbAo-preparation and of GBM of healthy children. The effects of non-enzymatic galactosylation of haemoglobin has been studied by high pressure liquid chromatography, the effects of GBM galactosylation by immunoelectrophoresis. Subsequently, the occurrence of elevated values for HbAIa-c and GSP was evaluated in 14 galactosaemic children (11 transferase deficiency, 3 galactokinase deficiency), as well as urinary acid glycosaminoglycae excretion and GBM immunoelectrophoretic mobility in 6 of these 14 children measured. The results were compared to the respective values of healthy control children. After exclusion of significant non-enzymatic glucosylation by measuring postprandial blood glucose values the galactosaemic children showed significantly increased values for HbAIa-c (8.85 +/- 2.0% versus 7.7 +/- 0.3%; p less than 0.02), for GSP (0.43 +/- 0.13 mmol 5-HMF/mg protein versus 0.32 +/- 0.07 mmol 5-HMF/mg protein; p less than 0.005) as well as for urinary acid glycosaminoglycane excretion (45.3 +/- 23.4 micrograms/mg kreatinine versus 9.9 +/- 2.3 micrograms/mg Kreatinine; p less than 0.01). 3 out of the 6 children showed alpha 1-immunoelectrophoretic mobility of GBM antigens which was found also after incubation of GMB with galactose. The other 3 children had alpha 2-immobility, which was found in the healthy controls as well as in the control incubations. The impact of galactose on increased non-enzymatic glycosylation in children with galactosaemia as well as the significance of this finding for diagnostic purposes or for clarifying pathophysiological aspects of the disease remains to be studied further.

Structural changes of hair after incorporation of the proline analogue L-azetidine-2-carboxylic acid. A model of hair disease by alteration of primary structure.

In order to correlate biochemical changes of the hair with physical properties we present a model for the examination techniques. L-azetidine was incorporated into the hair keratin complex and the resulting mechanoelastic properties were determined using the ultramicrohardness testing system on scanning electron microscopy. Structure was investigated by X-ray diffraction and incorporation of L-azetidine was detected by thin-layer chromatography. This system could possibly be introduced for examination of hair changes in humans. 8 white mice, 3 weeks of age, were given L-azetidine-2-carboxylic acid in water (0.1 g/100 ml) as only source of fluid over a period of 5 weeks. They had free access to dry mouse cake only. 8 animals of the same strain, who had free access to tap water and mouse cake and were kept under the same conditions, served as controls. After 5 weeks, the animals were sacrificed and hair was obtained for analyses. 2 dimensional thin-layer chromatography of hair hydrolyzed with 6N HCl at 105 degrees C for 12 hours revealed 2 additional spots in the chromatographic pattern in the experimental animals in comparison with the control group. 1 of the spots was identified as L-azetidine-2-carboxylic acid, while the second spot was possibly a degradation product of L-azetidine on acid hydrolysis at a high temperature. Hair of the animals was put into Mark capillaries and subjected to X-ray diffraction, which showed a markedly disordered orientation of keratin. Impression studies using scanning electron microscopy revealed a remarkably reduced elasticity of hair with incorporated L-azetidine. These findings may be explained on the basis of qualitative or quantitative changes in the helical structure of the keratin complex of hair, which is responsible for the elastic properties, whereas the globular matrix is responsible for the firmness of the hair.

[Effect of premature birth risk and prenatal care on the maturity and morbidity of the newborn infant]. / Einfluss von Frühgeburtsrisiko und Schwangerenbetreuung auf Reife und Morbidität der Neugeborenen.

Risk of premature birth was evaluated according to the prematurity risk score proposed by Thalhammer 1973 in 610 newborn infants hospitalized during 1974 to 1979 at the Division of Neonatology and Congenital Disorders of the Department of Pediatrics, University of Vienna. 324 infants had a birth weight of less than 2501 grams and 286 infants a birth weight of more than 2500 grams. Prematurity risk was compared with regard to prenatal care to birth weight and gestational age as well as to the duration of hospital stay, the incidence of respiratory distress syndrome, the need of ventilatory support and the mortality rate. Quality of prenatal care was judged from the frequency of medical attendances obtained during pregnancy. Less than 0,5 medical visits for 4 weeks were classified as bad prenatal care, more than 0,8 visits as good prenatal care. At any prematurity risk newborn infants from pregnancies with good prenatal care had a higher gestational age and a higher birth weight. They also had shorter hospital admissions, less frequent a respiratory distress syndrome or the need for ventilatory support and a lower mortality rate. The benefit of good prenatal care was supported further by the finding that important individual risk factors as well as the total prematurity risk score was the same in infants without respiratory distress syndrome but birth weights below 2501 grams as well as in infants with the respiratory distress syndrome in infants with ventilatory support and in infants who died. The mothers on the other hand of the healthy newborn infants weighing below 2501 grams shared significantly more medical attendances during pregnancy.

Urinary excretion of glomerular basement membrane antigens in premature infants and the newborn.

25 premature infants, 8 mature newborns and 25 children between 5 and 15 years of age were examined for urinary excretion of glomerular basement membrane (GBM) antigens. For the characterization of the excreted GBM antigen, immunoelectrophoresis was applied. In the group of 25 premature infants 23 showed alpha-1-mobility, in the group of 8 mature newborns all showed alpha-1-mobility, and in the group of the 25 children aged 5--15 years 24 showed migration into the alpha-2-zone. Differentiating, whether the difference between the immature and mature GBM is quantitative or qualitative, the immunoelectrophoretical difference points to the interpretation that the premature GMB shows a unique chemical composition.

Immunochemical characterization of mature and immature glomerular basement membrane.

20 kidneys from premature infants (27th to 38th weeks of gestation), 5 kidneys of mature newborns and 5 kidneys of children between 5 and 15 years of age were obtained at necropsy and glomerular basement membranes (GBM) isolated. The isolated GBMs were degraded by papain and the degradation products were characterized by immunoelectrophoresis applying an antihuman GBM antiserum from the rabbit. GBMs of children between 5 and 15 years showed in each case 3 precipitation lines distributed from the alpha-gamma-zone. The examined newborns and 17 premature infants presented two precipitation lines only, moving with alpha 1-mobility and beta-gamma-inter-region. 3 premature infants showed a pattern with 2--4 different precipitation lines of different mobility, maybe interpretable as a result of bacterial digestion. On the grounds of these findings we postulate that the GBM is, from an immunochemical point of view, immature at birth and in the late fetal life becoming mature in the child with about 5.

[The effect of collagenase inhibition on bullous eruption and healing process in epidermolysis bullosa]. / Einfluss von Kollagenasehemmung auf Blasenbildung und Heilungsverlauf bei Epidermolysis bullosa.

The collagenase inhibiting effect of erythromycin already observed in vitro was demonstrated also after oral administration of the drug in vivo in one child with epidermolysis bullosa letalis and one child with epidermolysis bullosa dystrophica. Despite inhibition of skin collagenase activity during administration of the drug the frequency of bullous eruptions and healing process of affected skin areas remained unchanged. This suggests no direct causal relation existing between skin collagenase activity and epidermolysis bullosa. Whether increased collagenase activity reflects a secondary reaction of the organism cannot be concluded from this study.

[Massive air embolism complicating artificial ventilation (author's transl)]. / Massive Luftembolie als Komplikation kontrollierter Beatmung.

A 1740 g premature infant being treated by intermittent positive pressure ventilation with PEEP because of respiratory distress syndrome died from massive air embolism occuring in two attacks on the 14th and 16th day of life. The pathogenesis of systemic air embolism in neonates with respiratory distress syndrome is discussed and the difficulties in diagnosis as well as the importance of this complication regarding possible cerebral damage are considered. There is no conclusive information about the incidence of this serious event available at present.