Programmed appearance of translatable flagellar tubulin mRNA during cell differentiation in Naegleria.

The programmed de novo synthesis of flagellar tubulin during the hour-long differentiation of Naegleria gruberi from amoebae to flagellates is our paradigm for the study of gene expression during cell differentiation. This paper reports the efficient translation of flagellar tubulin mRNA in the wheat germ cell-free system directed by total or polyadenylated RNA extracted from differentiating cells. The tubulin in the in vitro product has a subunit molecular weight of 55,000, separates into alpha and beta subunits under suitable conditions of polyacrylamide gel electrophoreis and co-polymerizes with calf brain tubulin. At least half of the tubulin synthesized in vitro is precipitated by antibodies specific to flagellar tubulin, and the immunoprecipitated tubulin subunits yield peptide maps similar to those of outer doublet tublin. Flagellar tubulin is the predominant protein synthesized in the cell-free system, and amounts to about 5% of the polypeptides whose synthesis is directed by total RNA from differentiating cells. In contrast, little or no flagellar tubulin is synthesized when the cell-free system is directed by RNA extracted from amoebae prior to differentiation. Translation assays show that at least 92% of the flagellar tubulin mRNA appears during differentiation. The time course of appearance of this mRNA was measured by quantitative immunoprecipitation of the cell-free products. Under conditions where cells from flagella 60 min after initiation of differentiation, translatable flagellar tubulin mRNA was first detected at 20 min, reached a maximum at about 60 min and then declined. An excellent correlation was observed between the amount of translatable flagellar tubulin mRNA and the previously measured rates of flagellar tubulin synthesis in vivo. These results indicate that synthesis of flagellar tubulin is a direct reflection of the abundance of its mRNA, and provide the molecular techniques for dissection of the factors that regulate the rapid appearance of this structural protein during differentiation.


Methylated and unmethylated DNA compartments in the sea urchin genome.

Sea urchin (Echinus esculentus) DNA has been separated into high and low molecular weight fractions by digestion with the mCpG-sensitive restriction endonucleases Hpa II, Hha I and Ava I. The separation was due to differences in methylation at the recognition sequences for these enzymes because an mCpG-insensitive isoschizomer of Hpa II (Msp I) digested Hpa II-resistant DNA to low molecular weight, showing that many Hpa II sites were in fact present in this fraction; and because 3H-methyl methionine administered to embryos was incorporated into the high molecular weight Hpa II-, Hha I- and Ava I-resistant fraction, but not significantly into the low molecular weight fraction. The fraction resistant to Hpa II, Hha I and Ava I amounted to about 40% of the total DNA. It consisted of long sequence tracts between 15 and well over 50 kg in length, in which many sites for each of these enzymes were methylated consecutively. The remaining 60% of the genome, (m-), was not significantly methylated. Methylated and unmethylated fractions were considered to be subfractions of the genome because enriched unique sequences from one fraction cross-reassociated poorly with the other fraction and specific sequences were found in either (m+) or (m-) but not in both (see below). Similar (m+) and (m-) compartments were found in embryos, germ cells and adult somatic tissues. Furthermor, we found no evidence for changes in the sequence composition of (m+) or (m-) between sperm, embryo or intestine DNAs, although low levels of exchange would not have been detected. Using cloned Echinus histone DNA, heterologous 5S DNA and ribosomal DNA probes, we have found that each of these gene families belongs to the unmethylated DNA compartment in all the tissues examined. In particular, there was no detectable methylation of histone DNA either in early embryos, which are thought to be actively transcribing the bulk of histone genes, or in sperm and gastrulae, in which most histone genes are not being transcribed. In contrast to these gene families, sequences complementary to an internally repetitious Echinus DNA clone were found primarily in the methylated DNA compartment.


Characterization and transcription analysis of a cloned sequence derived from a major developmentally regulated mRNA of D. discoideum.

The plasmid pDd 812 contains a portion of a poly(A)+ RNA sequence isolated from developing cells of the cellular slime mold Dictyostelium discoideum (Williams and Lloyd, 1979). The poly(A)+ RNA complementary to this plasmid shows an increase in concentration during the first 4 hr of development followed by a decrease in concentration during the following 4 hr. This RNA is very abundant after 3-4 hr of development, constituting at least 2% of the poly(A)+ RNA population. In this study, we demonstrate that this poly(A)+ RNA is an mRNA sequence by translating the RNA complementary to pDd 812 in a heterologous system. The mRNA directs the synthesis of a major polypeptide of 33,000 daltons and a minor polypeptide of 31,000 daltons. We have used the plasmid DNA immobilized on filters to analyze the transcription of this RNA sequence in isolated nuclei. The amount of transcript synthesized in nuclei isolated at various stages of development which was complementary to pDd 812 changed in the same way as did the cytoplasmic concentration of this RNA--that is, maximal transcription occurred after 3-4 hr of development. Because this result was observed using labeling periods as short as 5 min, we believe that this change is unlikely to reflect a change in the rate of processing of RNA. We interpret these results to indicate that, at least in part, the control of the synthesis of this RNA is at the level of gene transcription.


Location of additional early gene sequences in the adenoviral chromosome.

Genetic and biochemical analysis has revealed additional early gene sequences in the group C human adenovirus genome. Physical mapping by an improved marker rescue method showed that a group of "early function" mutations previously found by us to map within the left quarter of the genome do not map within the known early sequence (0-11.2) in that region, but instead lie within the coordinates 18.5-22.0. A search for early expression in this region of the genome using saturation hybridization techniques showed the presence of two discrete sets of viral cytoplasmic RNA sequences, present early in infection at low copy number and complementary to sequences on the I strand of the adenovirus genome lying between 11.2-14.5 and 19.8-23.5 untis.


Conditional expression of the transformed phenotype in an "epithelial-like" cell line.

A subclone of ts 14/MNU/2, derived from a heteroploid (epithelial) monkey kidney cell line of BSC-1 and transformed in vitro by methylnitrosourea, expressed the transformed phenotype only at the elevated temperature of 39.5(0)C. Compared to the growth characteristics at 33(0)C, the transformed property was exhibited in the (1) high efficiency of plating at low cell densities, (2) colony morphology, (3) growth in absence of serum and (4) alterations in the quality and the quantity of chromosomal proteins in presence of low concentrations of serum. Such experimental systems may provide an effective control for studying molecular mechanisms related to transformation and neoplasia.


Evaluation of a biochemical method for measuring platelet life-span.

Acetylsalicylic Acid (Aspirin) inhibits the formation of Malonaldehyde, a degradation product of the proaggregating Prostaglandins, during the life-span platelets in the circulating blood. After ingestion of 1.5 g of aspirin, there is a blockage of the formation of Malonaldehyde, followed by a progressive return to normal values, after an average of 8 days, in a healthy person. This fact is applied to the determination of half-life, found to be 3.7 +/- 1.3 days; a value in accord with those found by the isotopic method using 51Cr. The reproductibility of this method indicates a clinical application.


A three-dimensional study of organelle interrelationships in regenerating rat liver. 3. Organelles related to bile.

A number of cell structures are described which show a morphological relationship to the bile canaliculi. Two types of peribiliary vesicles are identified: osmication positive ones occurring between the bile canaliculi and the osmicated immature Golgi cisternae and probably deriving from the latter, and osmication negative ones related to MVB, on which they appear as buds. Small coated vesicles are seen attached to this second type. Large lacunae may originate from MVB, as suggested by the MVB-like internal vesicles they may contain. Some stay in luminal continuity with the bile canaliculi. Canalicular coated vesicles are seen as parts of the canalicular plasma membrane and free in the cytoplasm.


A three-dimensional study of organelle interrelationships in regenerating rat liver. 4. Multivesicular bodies.

Double-membraned plasma-membrane-loops are formed from the plasma membrane and released into the cytoplasm. The vesiculation of mainly their inner membrane may transform them into MVB. The so-formed internal vesicles contain cytosol. In many MVB the surface of all their internal vesicles to gether corresponds well with the surface of their bordering membrane. This correlation may be the result of a dynamic equilibrium whereby degradation (and partial recyclage) of internal vesicles is compensated for by the formation of new internal vesicles. When this equilibrium becomes disturbed, MVB may show much less internal vesicles. Such 'depleted' MVB are often producing peribiliary vesicles and may themselves transform into lacunate emptying into the bile canaliculi.


[Traumatic injuries of the epiphyseal plate in the lower extremity of the tibia].

In a series of 110 fractures of the ankle in children the authors have observed 27 cases of "at risque" lesions: Mac Farland types of fractures and crush injuries. Epiphysiodesis occurs in all cases of crush injuries and in 25% of fracture-separation of the tibial malleolus. In order to prevent this complication the only procedure is to obtain an anatomical reduction. Desepiphysiodesis is uncommunly possible (2 cases) and rarely effective. In case of varus deformity an osteotomy must be performed and repeated if the child is still young.


[Recurrent and multiple pheochromocytoma (author's transl)].

One case of multiple pheochromocytoma with several unusual features is reported: 1 -- the multiple localizations which came on at ten years interval. 2 -- Association with renal artery stenosis. 3 -- Association with Von Hippel Lindau's disease. The authors comment on this case and those from the literature.


[Is conservative treatment justified in the case of advanced infantile tuberculosis of the hip? (author's transl)].

In the case of advanced children tuberculosis of the hip, the authors propose a more active treatment than Wilkinson's, which is less conservative. A local curetting is important, and if the acetabulum reveals a geodic aspect, this surgery may be done by pelvic approach. After an orthopedic traction, the surgical method is more efficient and permits better flexibility of the hip, only to be noticed under general anesthesia. After surgery, this traction must be prolonged for a several months and kinesitherapic mobilisation must be associated. The excellent result, both clinical and radiographic obtained by the authors seems to justify the use of the above mentioned method.


[Disinsertion of the subscapularis muscle in the treatment of obstetric paralysis of the upper limb (author's transl)].

Proximal disinsertion of the subscapularis is an effective operation in the correction of an abnormal position in internal rotation of the shoulder. Such a decision should be taken only if the head of the humerus is still well rounded. Restoration of movement by muscular transfer of active external rotation is performed in subsequent months only if the teres minor and infraspinatus muscles do not recover spontaneously.


[Growth disturbance of the forearm in multiple exostosis and dyschondroplasia (author's transl)].

Multiple exostosis and Dyschondroplasia (Ollier's disease) are two Osteochondrodysplasia with abnormal cartilagenous growth which hinder growth of the long bones especially. Among other locations, these diseases do not affect the forearms symetrically. The ulnar bone is more often and more severely affected which results in a progressive dislocation of the forearm. The radial shaft bands while the hand incurves medially. The radial head can even dislocate completely. All the deformities of the forearm create a severe handicap. The authors report their surgical experience. Preventive management is the ideal solution and should be carried out before the onset of a complete dislocation (especially before the dislocation of the radial head). Correction should also take growth into consideration. Logically, the shortest bone (the ulnar) should be lengthened and straightened. Since it is not feasible to reasonably correct all deformities entirely by the ulnar lengthening, it is necessary to not only straighten but also to shorten the radial bone. Only regular surveillance of the growth abnormalities throughout childhood can allow a precise evaluation of the required correction necessary at the time of surgery which should be a definite one stage procedure.


[The surgical treatment of pectus excavatum (author's transl)].

The Authors who have operated on 120 cases of pectus excavatum, describe their own method of surgical correction. It consists of: -- Sternal mobilisation by sternotomies; -- Limited chondreal resections; -- Internal fixation. The Authors emphasize the interest of a good internal fixation which must allow normal breathing during the period of cartilage's strengthening. The use of long flexibles thread pins affords a good stability and flexibility and assures at long term good cosmetic results.


[Cricopharyngeal achalasia, exceptional cause of dysphagia in newborn (author's transl)].

Dysphagia of the newborn and the infant presents with many varied causes, neurological, malformative, turmoral or functional. Among the latter, cricopharyngeal achalasia is an exceptional entity open to discussion. Suspected as early as the first month, presenting with difficulty in swallowing with choking episodes and coughing up of food without any obvious etiology. Profile roetgengraphic studies will allow the diagnosis to be made. The characteristic sign is a round and regular posterior narrowing on the posterior wall of the esophagus (C4 - C5 level) with enlargement of the hypopharynx. This narrowing is a constant finding. This pathological entity, an absence of relaxation of the cricopharyngeus muscle during the third period of swallowing, well-known in the adult patient, has only exceptionally been described among newborn children. In the following case study, cure has only been obtained by an extra mucosal myotomy of the esophagus at the age of seven months. The discussion, considering the five cases reported in pediatric journals, covers the diagnostic elements, the physiopathology of the disease, the risks presented during evolution without treatment. Therapy must be precocious and surgical.


[Ovarian cysts and tumors in neonates, infants and children (author's transl)].

Twenty-six girls, aged from 0 to 15 years, were operated, upon ovarian cysts and tumors from 1952 to 1977. Before the age of one year, 11 non-neoplastic cysts and 2 benign tumors were observed. After the age of one, 1 non-neoplastic cyst and 12 tumors: 8 germ-cell tumors, 3 tumors of specialized gonadal stroma, and one epithelial tumor. Three germ-cell tumors were malignant (1 girl dead of disease, 2 alive at 2 and 1 1/2 years). All three gonadal stromal tumors are benign (with a follow-up of 3 months, 4 and 20 years). Epidemiology, pathology prognosis and management of these tumors are discussed according the recent literature.


[Renal lesions in children with acute traumatic abdominal bleeding (author's transl)].

In 22 cases we observed the association of post traumatic intra and retro peritoneal bleeding the latter due to kidney rupture. In such cases the usual policy consists in treating the abdominal visceral lesion first and post pone the management of the kidney rupture. In 13 cases we decided to undertake simultaneous surgical treatment of the abdominal and kidney lesion. The preoperative diagnosis was established by combination of exploratory peritoneal dialysis and I V P. The surgical exploration was achieved by means of medial incision. In 6/13 cases it disclosed kidney lesions which necessitated immediate nephrectomy. Our experience shows that one stage surgical procedure is beneficial to the patients.


[Synovial chondromatosis in children (author's transl)].

A serie of four patients is reviewed. We conclude that synovitis chondromatosis is very rare in children; the diagnosis, accepting Jaffe definition is not easy. In this serie age and localisation on the hip modify consequently our indication of the treatment, and explain some recurrence.


[The epiphyseal defect in children (author's transl)].

The authors produced four cases which all show at first almost the same radiographical espect: an epiphysal defect of the knee. The first case developed in an inflammatory context and proved to be an osteomyelitis. The second case showed a begnin tumour on X-Ray examination a chondroblastoma. Then an epiphysal tuberculosis was produced which at first led to a rather unsettled diagnosis. The last case was a rare observation of chondrome. The diagnostic problems are frequent and besides possible infections localisations, the diagnosis of chondrablastome seems to be the most likely. Surgical approach of such epiphysal lesions is justified not only to corroborate etiology but also in order to avoid an increase in the volume that could impair the epiphysal plate endanger the growth.


[Brachial plexus birth injuries. An experimental study (author's transl)].

The authors present an experimental study of obstetrical Brachial plexus Palsy. They study traction injury to 16 Brachial pleux. The first lesions were always disruption of the upper roots C5 and C6. After that, the Lower Plexus (C7 - C8 - T1) was disrupted. The two upper roots are usually interrupted and surgical graft seen to be possible. On the Lower Plexus, avulsions are found and intercostal nerves transfer must be performed.


[Xanthogranulomatous pyelonephritis in the infant. A report on 4 cases (author's transl)].

Four cases of xanthogranulomatous pyelonephritis in children are reported. In three cases, the disease took a diffuse form and in the fourth it was of a localised form. Around these cases, the authors review 39 case histories published between 1963 and 1978 and which involve only children. An over-all assessment of the 43 cases thus gathered together allows the general picture of xanthogranulomatous pyelonepn 9 cases, it was of the localised type; and in 4 cases, the pyelonephritis only involved one or another pyelon in patients with re-duplication. The diffuse type is more common in boys and the localised type in girls. Twenty-nine cases were found in children of less than five years. In 23 cases, there was a proteus infection. The etio-pathogenesis of xanthogranulomatous pyelonephritis remains obscure. The main clinical and therapeutic problems are dealt with.


[Endoscopical measures of length of intramural ureter in children with primary vesico-ureteral reflux (author's transl)].

153 endoscopical measures of the length of the intra-mural ureter in 81 children over 2 years have enabled us to ascertain that this segment of vesical ureter is significantly shorter in most patients with vesico-ureteral reflux. By contrast with other previous studies, we have not been able to use this examination for establishing the prognosis of the reflux, perhaps by lack of sufficiently prolonged follow-up However these data have been helpful to us in two circumstances and were complementary of the determination of aspect and position of ureteral meatus: a - When the child is operated for au unilateral reflux, a controlateral short intra-mural ureter suggests the value of a bilateral reimplantation to avoid the appearance of a controlateral reflux. b - When the child has an urinary infection without demonstrated reflux, a short intra-mural ureter suggests the value of a repeated cystography.


[Constipation in the infant (author's transl)].

The authors studied 102 cases of constipation in children. 76 of them went through a protocol of investigations : clinical, psychological, radiological and manometric. The authors suggest that so-called functional constipation is more often organic than is commonly thought.


[The elasticity co-efficient of the rectal wall (E.C.R.P.). A prognostic test for Hirschsprung's disease (author's transl)].

In a situation where all authors are agreed on the significant morbidity of a colostomy in Hirschsprung's disease, the new test described by the authors would appear to help, if not "resolve" the always difficult choice between a colostomy and simple nursing in the intermeditary treatment of Hirschsprung's disease of the newborn.


[Wiedemann-Beckwith syndrome heredity pattern (author's transl)].

The authors report a sibship of four members (two male and two female)displaying the entire clinical picture of the Wiedemann-Beckwith syndrome. There was no consanguinity and no antecedents could be found in a three-generation search. On this occasion, a review is made of what is known about the Wiedemann-Beckwith syndrome concerning its hereditary pattern, which can currently be considered as autosomal dominant with variable expressivity and incomplete penetrance, instead of recessive as it was formerly accepted This heredity pattern must be known in order to provide an accurate genetic advise.


[Apple peel small bowel. Report of two cases of a peculiar type of small bowel atresia (author's transl)].

The authors report two personal cases of a peculiar type of small bowel atresia called "Apple peel small bowel", they give proheminence on the unusualiness of the lesion, the non specific clinical picture, and the relatively bad prognosis. The physiopathologic hypothesis which were proposed for this disease are reviewed.


[Segmental dilatation of the ileum associated with an omphalocele and a common mesentery. Discussion around 2 cases : chance or morphogenically "related"? (author's transl)].

Segmental dilatation of the ileum is the subject of this report which concerns two cases. Both cases were associated with omphalocele and intestinal malrotation, an observation already noticed by some authors. Is it still a hazardous coincidence or rather a real opportunity which throws light over the explanation of this malformation?


[Strangulation of the toes by hair (author's transl)].

The purpose of this report is to call attention to human hair as a potential source of injury to young children's toes. The toe tourniquest syndrom is an entity described by Quinn but the autors suspected intentional wrapping.


[Incontinence after repair of imperforate anus : treatment by a free muscle transplant (author's transl)].

A case of severe anal incontinence after multiple surgical repairs for ano-rectal imperforation is presented. A successful formation of a new puborectalis sling by means of free autogenous palmaris longus muscle transplantation according to Hakeliuis is reported. The clinical success is confirmed radiologically and electromyographically. The authors discussed the principles and the results of this new method. They believe that this case pleads in favour of true muscle autotransplant re-innervation.


[The childs-philips intestinal plication. A long term follow-up of 11 patients (author's transl)].

The authors report a long term follow up of 11 children who underwent a Childs-Philips intestinal plication from one to fourteen years ago. One death was caused by this operation. Besides, the tolerance of the Childs-Philips operation seems to be good, the nine other patients present a normal growth without any recurrence of the occlusion. The authors emphasize the interest of this bowel plication in the treatment of occlusion during the childhood.


[The place of lavage-irrigation of the peritonium in peritonitis from the appendix in children. A report of 10 cases (author's transl)].

Confronted with the difficult problems presented in acute appendicular peritonitis, whether generalized or multiple focused, the authors used the "lavage irrigation" method in ten cases. They analysed the local and general conditions that led them to employ this method, in light of the evolution of this technic which they had observed in their experiences whit it. They have attempted to elucidate, using information previously available in the literature, the indications, the steps, and the eventual complications of the "lavage irrigation" of the peritonemum in the child.


[End to side porto-caval shunt in familial type II homozygote hypercholesterolemia (author's transl)].

Two patients with homozygous type II familial hypercholemia have had significant improvement by end to side porta-caval shunt. Follow-up is respectively 1 and 4 years. When all the other treatment have failed in lowering blood cholesterol and L.D.L. levels, a portal diversion may help to prevent cardio-vascular atherosclerotic complications in the pediatric age.


Time course of increased collateral arterial and venous endothelial cell turnover after renal artery stenosis in the rat.

We induced left renal artery stenosis in rats and studied collateral arterial formation by angiography, histology, and radioautography with tritiated thymidine. Endothelial cell turnover was estimated by radioautography with tritiated thymidine in the periureteric blood supply of 10 normal and 38 collateral-forming kidneys 1 to 100 days after stenosis. Periureteric arterial endothelial cell labeling showed a highly significant (P less than 0.005) increase, apparent within 1 day and gradually falling as the vessels grew, until a baseline was reached in 35 days. A smaller but statistically significant increase in the labeling index also was found in endothelial cells of the renal vein during the first week (P less than 0.01), and had a similar time course. A marked increase in epithelial cell labeling in the ureters draining the stenotic kidneys also was evident (P less than 0.005). Thus, collateral vessel development is characterized by active DNA synthesis in the cellular elements which is maximal during the first week. A humoral factor is implicated in the vascular response by the parallel proliferation of venous and uretic cellular elements that are unlikely to experience the biophysical forces, such as increased blood flow or tangential wall force, which might stimulate proliferation in ther arterial vessels.


Disturbed maternal bereavement following infant death.

This report describes mental health intervention with the mothers of children with behavioural disturbances following infant death. Disturbed maternal bereavement disrupted the mother's relationships with their own children and related to maladaptive handling of prior losses. Treatment directed toward the recovery of memories and effects related to these losses led to more adaptive parent-child relationships. The implications for preventive interventions with families are discussed.


Congruence of childhood sex-role identity and behaviour disturbances.

Thirty-eight children who were aged between 4 and 12 1/2 years were referred for potential gender disturbances and received independent assessments of sex-role behaviour and identity. Ratings of sex-role behaviour were based on psychodynamic, behavioural and psychometric procedures, with the behavioural and psychodynamic ratings most highly correlated. Two ratings of sex-role identity disturbance, derived from psychodynamic and behavioural orientations, yielded a strong interrelated agreement. The mean ratings of sex-role identity and behaviour disturbance were moderately correlated and provided a high degree of internal consistency. An overall diagnosis was derived for each subject by combining the behaviour and identity mean ratings. The findings are discussed in terms of the limitations of present knowledge on normal and deviant sex-role developmental processes.


Effects of neural stimuli on blood flow through vasa vasorum in dogs.

The purpose of this study was to determine whether neural stimuli alter blood flow through vasa vasorum in the thoracic aorta. We measured flow with microspheres in anesthetized dogs and observed responses to sympathetic stimulation and baroreceptor stimulation. During these interventions, changes in arterial pressure were prevented with propranolol or an arterial reservoir, to minimize indirect effects on vasa vasorum mediated through changes in aortic wall tension. Electrical stimulation of the stellate ganglion at 10 Hz reduced blood flow to vasa vasorum in the thoracic aorta from 11 +/- 1.6 (mean +/- SE to 6.8 +/- 1.1 ml/min per 100 g (p less than 0.05). Aortic diameter, measured with a sonomicrometer technique, did not change during sympathetic stimulation. Thus, the reduction in blood flow through vasa vasorum during sympathetic stimulation appears to be a direct effect, and not the result of constriction of the aorta and compression of vasa. To determine effects of physiological alterations in neurogenic vasoconstrictor activity, we examined responses to stimulation of carotid baroreceptors. When pressure in isolated, perfused carotid sinus baroreceptors was raised from 81 +/- 3 to 198 +/- 2 mm Hg, blood flow to vasa vasorum of the thoracic aorta increased from 3.7 +/- 0.6 to 10 +/- 2.2 ml/min per 100 g (P less than 0.05). We conclude that vasa vasorum are responsive to neural stimuli, since they constrict during sympathetic stimulation and dilate in response to baroreceptor stimulation.


The plasma membrane of leading pacemaker cells in the rabbit sinus node. A qualitative and quantitative ultrastructural analysis.

We studied the fine structure of the plasma membrane of electrophysiologically identified leading pacemaker cells from the rabbit sinus node, using both ultrathin sections of fixed tissue and replicas of freeze-cleaved material. We found that differences exist between sinus node and working myocardial membranes, but these are only quantitative. The caveolae or sarcolemmal invaginations are present in very large numbers; they increase the surface area of the plasma membrane by about 100%. The small macular nexuses that are present represent 0.2% of the membrane surface area. Nexuses are therefore about 10 times less numerous in leading sinus node cells than in working myocardium cells. A single equivalent electrical representation of the sinus node shows, nevertheless, that an appreciable electrical coupling may be expected.


Modulating effect of regional myocardial performance on local myocardial perfusion in the dog.

We studied the effect of regional contractile performance on regional coronary blood flow and flow distribution in 10 dogs. The left anterior descending (LAD) coronary artery was cannulated and perfused. Maximal vasodilation was obtained with adenosine. Consequently, variations of LAD flow reflected changes of extravascular resistance. Lidocaine injected in the LAD caused a localized reduction of contractile performance as shown by the absence of systolic wall thickening. Global left ventricular performance and pressure were unchanged. Coronary extravascular resistance diminished and LAD flow increased from 4.8 +/- 0.5 to 6.2 +/- 0.6 ml/min per g (P less than 0.02). The endocardial: epicardial ratio increased from 1.02 +/- 0.07 to 1.28 +/- 0.07 (P less than 0.001). Isoproterenol in the LAD augmented systolic wall thickening. Regional coronary flow diminished from 5.1 +/- 0.5 to 3.3 +/- 0.4 ml/min per g (P less than 0.001), and the endocardial:epicardial ratio diminished from 1.08 +/- 0.07 to 0.75 +/- 0.07 (P less than 0.01). These data indicate that myocardial contractility is a major component of extravascular coronary resistance and is a mechanical determinant of coronary blood flow and its transmural distribution.


Cellular thin filament protein contents and force generation in porcine arteries and veins.

We estimated the cellular myosin, actin, and tropomyosin contents of vascular smooth muscle from (1) seven major arteries, (2) seven large veins, and (3) the first through third order branches of the uterine vasculature to determine whether variations in the contractile apparatus contribute to the functional diversity of vascular smooth muscle. We obtained the estimates by quantitative densitometry of stained polyacrylamide gels after electrophoresis of sodium dodecyl sulfate-treated tissue homogenates. No differences in cellular myosin content were found (18.7 +/- 1.0 mg/g cell wet weight in arteries vs. 17.2 +/- 0.7 in veins). However, the actin and tropomyosin contents were higher in arteries (49.7 +/- 2.9 and 13.1 +/- 0.8 mg/g cell, respectively) than in veins (25.5 +/- 1.4 and 7.0 +/- 0.3 mg/g cell). These differences persisted in the smaller uterine vessels. The higher contents of thin filament proteins in arteries, compared with veins and several other smooth muscle tissues previously studied, may underlie the high force generating capacity of arterial smooth muscle.


Dynamic changes in the canine mitral regurgitant orifice area during ventricular ejection.

We designed this study to test the hypothesis that in acute mitral regurgitation the mitral regurgitant area (MRA) is a dynamic quantity which varies with the time variation of ventricular volume. Mitral insufficiency was created in five open-chest dogs in which a portion of the anterior leaflet was excised. Phasic aortic and mitral flows were measured electromagnetically, along with left atrial and ventricular pressures. Filling, regurgitant, and stroke volumes, and systolic pressure gradient were determined by digital methods. MRA was calculated from the fluid dynamic equation of motion to give the temporal mean and the instantaneous value at three instants of time and at the time of peak flow (when inertia is negligible). Mean regurgitant fraction was 42 +/- 12% with no indication of left ventricular failure due to volume overload. MRA decreased monotonically with time to 59% of its initial value and closely paralleled the decrease in ventricular volume during systole. In a control study using a tilting-disc prosthesis with a hole 5 mm in diameter in the occluder, the calculated MRA was time invariant and equal to the measured area for regurgitation. We conclude that in acute mitral regurgitation the MRA is a function of ventricular volume.


Mechanism of action of methyldopa in the rat. Role of 3-O-methylated metabolites.

We studied the effect of 3-O-methyl-methyldopa (OMMD), the 3-O-methylated metabolite of the antihypertensive drug methyldopa (alpha-methyldopa, AMD), on blood pressure in the spontaneously hypertensive rat. OMMD lowered blood pressure in a dose-related manner when given orally or intraperitoneally. Its action lasted longer than that of AMD, and daily oral administration produced a cumulative fall in blood pressure. Oral and intraperitoneal OMMD produced similar reductions of blood pressure and similar tissue OMMD levels. After intraperitoneal injection of different doses, levels of OMMD measured in brain, spinal cord, and plasma correlated with the magnitude of the antihypertensive effect. No AMD was detected in tissues after either route of administration, which suggests that the antihypertensive effect was not based on demethylation of OMMD to AMD. Peripheral inhibition of the enzyme, aromatic amino acid decarboxylase (AAAD), failed to suppress OMMD's effect on blood pressure; in contrast, central inhibition of AAAD did decrease OMMD's antihypertensive effect. These observations suggest that 3-O-methylated metabolites may participate in the antihypertensive effect of AMD.


Exercise-induced U-wave inversion as a marker of stenosis of the left anterior descending coronary artery.

The prevalence and cineangiographic correlates of exercise-induced inversion of U waves were studied in 248 patients. Exercise-induced U-wave inversion was observed in 36 patients (15%), of whom 35 had greater than or equal to 75% stenosis in one or more of the major coronary arteries. The proximal left anterior descending or left main coronary artery was involved in 33 of these patients, including 24 patients with no electrocardiographic evidence of anterior myocardial infarction. Exercise-induced U-wave inversion was observed in the absence of an abnormal resting 12-lead ECG. Only one of the 82 patients (1.2%) without significant coronary artery disease demonstrated exercise-induced U-wave inversion, and this patient had a primary cardiomyopathy. We conclude that exercise-induced inversion of the U-wave is highly predictiveof significant coronary artery disease and, more specifically, of disease of the proximal left anterior descending coronary artery.


Normal electrocardiographic waveform characteristics during treadmill exercise testing.

Forty asymptomatic male patients at low risk for cardiovascular disease completed maximal treadmill testing. Electrocardiograms from leads CC5, CM5, V5, Yh and Z were recorded across multiple pretest, exercise and recovery conditions. ECG waveforms were subsequently digitized, averaged and processed to provide Q-, R-, S- and T-wave amplitudes, ST-segment means and slopes, and QS- and RT-interval durations. Average R-wave amplitude increased during early exercise and then dramatically decreased to maximum effort. Average S-wave amplitude became greater as exercise progressed. Average J junction was slightly positive before exercise, became negative during exercise (except lead Z) and returned to zero after exercise. The ST-segment slope increased dramatically with progressive exercise. The response of T-wave amplitude, RT and QS intervals are also described. Separately, 22 asymptomatic male subjects each completed two maximal treadmill tests 2 weeks apart. ECG data acquisition and processing were similar to those noted above. Pooled, within-subject estimates of variability were computed for the ECG leads, ECG measurements and protocol conditions. These variability estimates are useful for interpreting ECG responses to exercise testing.


The electrocardiographic and antiarrhythmic effects of imipramine hydrochloride at therapeutic plasma concentrations.

The electrocardiographic effects of imipramine hydrochloride at therapeutic plasma concentrations were determined in 44 depressed patients during a 6-week clinical outcome study of depression. During each week of the protocol, i.e., 2 weeks of control and 4 weeks of drug treatment, a standard 12-lead ECG, high-speed, high-fidelity ECG tracings, and a 24-hour continuous ECG recording were obtained. PR, QRS, and QTc intervals, T-wave amplitude, heart rate and frequency of ventricular premature depolarizations (VPDs) were measured. The plasma concentration of imipramine and desmethylimipramine was measured three times a week. Imipramine prolonged the PR (p less than 0.001), QRS (p less than 0.001) and QTc (p less than 0.001) intervals, increased the heart rate (p less than 0.001) and lowered T-wave amplitude (p less than 0.05) during the 4 weeks of treatment. No patient developed high-grade atrioventricular block or severe intraventricular conduction abnormalities. In addition, imipramine had a potent antiarrhythmic action in patients who were recovering from depression. Ten of 11 patients who had more than 10 VPDs/hour had 90% or greater arrhythmia suppression during antidepressant treatment with imipramine at plasma concentrations ranging from 100--302 ng/ml.


Adverse hemodynamic effects of intravenous disopyramide compared with quinidine in conscious dogs.

Disopyramide resembles quinidine electrophysiologically, but its effect on left ventricular function has not been clarified. Twelve awake dogs were instrumented for measurement of cardiac output, left ventricular pressure and its maximal first derivative (dP/dt max), and left atrial and aortic pressures. Disopyramide or quinidine at identical, clinically relevant doses (1 and 5 mg/kg i.v.) was infused over 5 minutes at each level. Peak changes after disopyramide 1 mg/kg included increases in heart rate (34%), mean aortic pressure (24%) and systemic vascular resistance (33%), and decreases in stroke volume (16%) and dP/dt max (19%). With disopyramide at 5 mg/kg these changes were of greater magnitude (e.g., dP/dt -- 36%). Quinidine at both doses caused no changes except a 13% decrease in vascular resistance at 5 mg/kg. Heart rate with disopyramide increased after propranolol (1 mg/kg i.v.), was unchanged after atropine (0.1 mg/kg i.v.), and slowed after propranolol and atropine. Phenoxybenzamine (2 mg/kg i.v.) did not prevent the rise in systemic vascular resistance produced by disopyramide. Thus, disopyramide in clinical dosages exerts opposing direct and indirect effects on cardiac pacemakers and, unlike quinidine, is a potent myocardial depressant and vasoconstrictor in the conscious dog.


Histology of pulmonary arterial supply in pulmonary atresia with ventricular septal defect.

A histologic study was performed on 22 specimens of pulmonary atresia with ventricular septal defect to 1) ascertain the existence of the main pulmonary artery; 2) distinguish the ductus arteriosus from the systemic collateral arteries (SCA); 3) establish the nutritive or functional nature of collateral circulation; and 4) evaluate the morphology of the distal pulmonary bed. Three cases had absent main pulmonary artery, one with and two without signs of infundibular septation. We suggest that absent main pulmonary artery may exist with both infundibular pulmonary atresia and persistent truncus arteriosus. SCAs have been found to have similar histological features as systemic muscular arteries of the same size--their medial muscular layer merges gradually into an elastic one at different depth inside the lungs. Injection of contrast material allowed us to demonstrate that these vessels are functional, since they inosculate into efficient pulmonary arteries ending in the respiratory units. When the distal pulmonary vascular bed is perfused by large SCAs, proliferative lesions like those found in large left-to-right shunts may occur. Early in infancy, banding of large, nonstenotic SCAs could protect the distal pulmonary vasculature. Moreover, total surgical repair should be associated with ligation of the SCA to avoid residual left-to-right shunt, if the pulmonary arteries can carry the full pulmonary blood flow.


Effects of acute hemodynamic alterations on pulmonic valve motion. Experimental and clinical echocardiographic studies.

The purpose of this study was to assess the effects of acute alterations of the pulmonary circulation on the pulmonic valve echocardiogram. We measured the pulmonic valve opening velocity (PVOV) and right-sided systolic time intervals (right ventricular preejection period-to-right ventricular ejection time ratio [RPEP/RVET] in 22 open-chest dogs subjected to acute hemodynamic alterations produced by inferior vena cava constriction, atrial pacing, isoproterenol infusion and microsphere embolization of the pulmonary artery. We found only fair correlations between PVOV and peak pulmonary artery flow (r = 0.56), right ventricular dp/dt (r = 0.43), stroke volume (r = 0.42), pulmonary artery systolic pressure (r = 0.33) and peak pulmonary artery acceleration (r = 0.31). RVET was shortened by reduced venous return (caval constriction) and by increases in heart rate (atrial pacing and isoproterenol), which resulted in increases in RPEP/RVET that did not correspond well to simultaneous changes in pulmonary artery pressure. In seven patients breathing 10% O2 to raise pulmonary artery pressure acutely, we found no change or a fall in PVOV. Thus, the pulmonic valve echocardiogram is influenced by multiple factors relating to parameters of pulmonary flow and right ventricular contractility, and may be of limited clinical usefulness in predicting pulmonary artery pressures.


Noninvasive assessment of atrioventricular pressure half-time by Doppler ultrasound.

The mean pressure drop across the mitral valve and atrioventricular pressure half-time were measured noninvasively by Doppler ultrasound in 40 normal subjects, in 17 patients with mitral regurgitation, 32 patients with mitral stenosis and 12 with combined stenosis and regurgitation. In normal subjects pressure half-times were 20--60 msec, in patients with isolated mitral regurgitation 35--80 msec and in patients with mitral stenosis 90--383 msec. There was no significant change in pressure half-time with exercise or on repeat examinations, indicating relative independence of mitral flow. In 25 patients with mitral stenosis and seven with combined stenosis and regurgitation, pressure half-time was related to mitral valve area calculated from catheterization data. Increasing pressure half-times occurred with decreasing mitral valve area, and this relationship was not influenced by additional mitral regurgitation. Noninvasive measurement of pressure half-time together with mean pressure drop was useful for evaluating patients with mitral valve disease.


Cross-sectional echocardiography. I. Analysis of mathematic models for quantifying mass of the left ventricle in dogs.

Cross-sectional echocardiography was used to quantify left ventricular mass noninvasively in 21 dogs. Short- and long-axis cross-sectional images of the left ventricle were reproducibly traced at endocardial and epicardial borders during stop-motion video-tape replay. We used area, length and diameter measurements to calculate left ventricular mass by seven mathematic models, including the standard formulas used with M-mode echocardiography and cineangiography. Calculated mass was compared with excised weight of the left ventricle by regression and percent error analyses. Formulas using short-axis areas and long-axis length resulted in higher correlation coefficients (0.94--0.95) and lower mean errors (6--7%) than for standard formulas. Since short-axis areas account for regional left ventricular irregularities, noninvasive quantification of left ventricular mass by cross-sectional echocardiography in dogs is most accurate with formulas using short-axis areas.


Scintigraphic characteristics of experimental myocardial infarct extension.

Technetium-99m-stannous pyrophosphate scintiphotos were evaluated for diagnosing and quantitating myocardial infarct (MI) extension in sedated dogs. Infarction and extension were produced by serial left anterior descending coronary artery ligations at 0 and 48 hours. We compared serial scintiphoto data with regional myocardial blood flow (MBF) (microsphere technique) and infarct histopathology. In eight control dogs, the scintigraphic MI area was stable at 24, 48, and 72 hours. In each of 11 dogs undergoing extension, the MI area increased after the 48-hour occlusion, averaging a 48.9% increase (p less than 0.001). Grossly, most extensions were mixtures of confluent necrosis and moderate (patchy) necrosis. MBF to confluent infarct tissue decreased significantly, allowing the documentation of extension by totaling the grams of newly flow-deprived tissue, but patchy infarct tissue had little flow deprivation, making it difficult to quantitate this type of extension accurately by flow criteria alone. Rarely, extension could be diagnosed using conventional histologic criteria. We concluded that the scintiphoto MI area was related quantitatively to infarct weight in both control and extension. However, it was not possible to determine that an increase in the MI scintiphoto area was an accurate predictor of the degree of extension using independent flow or pathologic criteria.


Evaluation of transthoracic electrical impedance in the diagnosis of pulmonary edema.

To evaluate the clinical usefulness of measuring transthoracic electrical impedance in patients with pulmonary edema, we studied 27 normal subjects and 33 patients. In normal subjects, impedance increased when body position changed from supine to standing (p less than 0.01) and when lung volume increased from residual volume to total lung capacity (p less than 0.01). Impedance values vari7 omega) when measured hourly without removing the electrodes. In only five of 11 patients with clinically severe pulmonary edema and five of eight with radiologically severe pulmonary edema were impedance values outside the normal range. Changes in impedance values correlated with changes in clinical and radiographic indexes of pulmonary edema. We conclude that (1) single measurements of impedance are useless in diagnosing pulmonary edema, and (2) impedance changes as predicted as pulmonary edema increases and decreases, and these changes are readily detected by simpler methods of evaluation.


Two-dimensional echocardiographic demonstration of left atrial thrombi in patients with prosthetic mitral valves.

Although M-mode echocardiography (MME) is not a reliable method for detecting left atrial thrombi, recent reports suggest that two-dimensional echo (2DE) may be more effective than MME in identifying intracardiac thrombi. In three patients with prosthetic mitral valves who presented with either arterial embolization or prosthetic valvular dysfunction, 2DE demonstrated left atrial masses consistent with thrombi, while MME was either negative (two patients) or suspicious (one patient) for left atrial thrombus. Thrombi were documented by surgical or postmortem examination in all cases. Clear delineation of the atrial cavity and the margins of the masses, visualization on multiple echocardiographic views and comparison of serial examinations were helpful in identifying these masses as thrombi. In addition, the masses visualized had certain patterns of motion which seem unique and may allow characterization of atrial masses as thrombi.


Computer diagnosis of supraventricular and ventricular arrhythmias. A new esophageal technique.

Computerized arrhythmia monitors recognize only a few of the significant arrhythmias and generally fail to detect arrhythmias of supraventricular origin. This is because conventional surface leads, which are sufficient for QRS recognition, are highly inadequate for automated P-wave detection. A new two-lead system, which includes a swallowable capsule-electrode for esophageal monitoring of atrial activity, is used in an on-line arrhythmia monitor. Three interval measurements (AA, AR and RR) and a QRS shape measurement provide the foundation for a detailed interpretation of each beat. Building on the single-beat analysis, a contextual diagnostic algorithm then recognizes and reports on-line the following arrhythmias: couplets, bigeminy, trigeminy, ventricular tachycardia, supraventricular tachycardia, atrial flutter, atrial fibrillation, ventricular tachycardia with retrograde conduction to the atria, first-degree block, second-degree block, Wenckebach periodicity, advanced block, third-degree block and sinus bradycardia.


Ventricular tachycardia and ventricular fibrillation in a young population.

In this study, we describe the findings in 18 young patients (age range 4 days to 24 years, mean 16.6 years) who had ventricular tachycardia and/or ventricular fibrillation and were followed for 4--70 months (mean 22.4 months). Patients had a variety of problems associated with their arrhythmia, including mitral valve prolapse, cardiomyopathy, myocarditis, prolonged QT syndrome and hypokalemia. Six patients had no clinically recognizable cardiac abnormality. The ventricular tachycardia showed a left bundle branch block contour in 10 of 17 patients, right bundle branch block in four, was multiform in two and had an indeterminate contour in one. Sustained ventricular tachycardia was initiated and terminated reproducibly by atrial and ventricular stimulation in three of seven patients who did not have spontaneous episodes of ventricular tachycardia during the electrophysiologic study. In one other patient, short bursts of ventricular tachycardia were induced. Patients who had ventricular fibrillation, those who died, and those who are still symptomatic with poorly controlled ventricular arrhythmias had significant heart disease. In one patient, a ventricular tachyarrhythmia that had required more than 100 electrical cardioversions spontaneously disappeared after requiring 1 year of antiarrhythmic therapy.


Ventricular ectopic beats and their relation to sudden and nonsudden cardiac death after myocardial infarction.

The role of ventricular ectopic beats (VEBs) in identifying patients who die of cardiac cause in the posthospital phase of myocardial infarction was evaluated in 940 patients who survived an acute coronary event. Six-hour Holter ECG recordings were obtained before hospital discharge, and VEBs were classified as complex (bigeminal, multiform, repetitive or R on T), simple (one or more VEBs that did not have complex patterns), or not present. Patients were followed 1-60 months (average 36 months) and cardiac mortality was categorized as sudden (less than or equal to 1 hour) or nonsudden (greater than 1 hour) among 98 witnessed cardiac deaths. Complex VEBs were associated with a significantly increased cardiac death rate, but did not discriminate between sudden and nonsudden death. Simple VEBs were associated with a 3-year cardiac mortality rate intermediate between those with complex and those with no VEBs. The relationship between complex VEBs and cardiac mortality was independent of 10 relevant clinical variables.


Motilin, human pancreatic polypeptide (HPP) and gastrin plasma concentrations in fasting subjects.

Motilin, human pancreatic polypeptide (HPP) and gastrin plasma concentrations have been determined in 196 fasting subjects ranging in age from the third to ninth decade. Detailed descriptions of the motilin, HPP and gastrin radioimmunoassays are presented. Plasma motilin concentrations ranged from undetectable to 1253 ng/L with a median value at 157 ng/L. Age and sex did not influence plasma motilin concentrations. Plasma HPP concentrations ranged from undetectable to 1564 ng/L with a median value at 142 ng/L. Plasma HPP concentrations increased significantly with age; males generally had higher concentrations than females. Plasma gastrin concentrations ranged from undetectable to 1658 ng/L with a median value of 21 ng/L. Age and sex did not influence plasma concentrations. These data establish ranges for plasma motilin, HPP and gastrin in fasting subjects aged 21--90 years using the described radioimmunoassays.


Evaluation of serum digoxin and serum cortisol by radioimmunoassay with the Concept-4.

The solid-phase radioimmunoassay procedures for serum digoxin and cortisol with the Concept-4 were evaluated for specificity, correlation with other radioimmunoassay procedures, parallelism, recovery, carryover, and precision. 1. The detection limits of the digoxin and cortisol assays were 10 pg/tube and 40 pg/tube, respectively. These detections correspond to serum concentrations of 0.25 micrograms/liter and 20 micrograms/liter, respectively, for digoxin and cortisol. 2. Specificity of the digoxin assay was characterized by a 18.3% cross reaction with digitoxin. The anti-cortisol with 11-deoxycortisol, cross reaction with 11-deoxycortisol. 3. The values obtained with the Concept-4 for both procedures demonstrated acceptable correlations with other existing RIA procedures. 4. Dilution studies conducted to determine the parallelism of the 2 assays indicated that both assays demonstrated excellent proportionality between the volume of specimen and the quantity of analyte measured. 5. Analytical recoveries of digoxin and cortisol added to the individual assays average 95% and 101%, respectively. 6. Carryover studies indicated a routine sample-to-sample interaction of less than 0.05% for both assays. 7. The digoxin assay routinely demonstrated intra- and inter-assay variations of less than 7.4% and less than 10.0%, respectively. The precision data for cortisol indicated intra- and inter-assay variations of less than 6.7% and less than 9.1%, respectively.


Investigation of reaction intermediates of the urea-diacetylmonoxime reaction.

1. An investigation of the intermediates of the urea-diacetylmonoxime reaction and the isolation of the protochromogen has been described. A comparative spectral study of the protochromogen with that of the literature values suggested the structure of the protochromogen to be a diureide. 2. The diureides of 1-phenyl-1,2-propanedione-monoxime and 2,3-pentanedione were synthesized and their visible, U.V., I.R., NMR, and mass desorption spectrometry data were analyzed. The study suggests that the structure of each of these compound is that of a diureide. When these diureides were redissolved in acid media, they gave the color expected of the corresponding chromogens found from the respective diketones and urea. 3. The 3a-methyl-6a-phenyl-glycoluril was synthesized by a base-catalyzed reaction. When this glycoluril was dissolved in concentrated HCl, a pink color was obtained, similar to that of the corresponding diureide. These studies indicate that both the diureide and the glycoluril (if formed at all in acid-catalyzed reaction) are converted to the same chromogen, possibly via a rearrangement. 4. When 2,3-butanedionemonoximethiosemicarbazone was reacted with urea in acid medium, characteristic spectral data were obtained which were very similar to those of the urea-diacetylmonoximethiosemicarbazide reaction. This tends to indicate that the possible structures of the protochromogens are similar and of the open chain diureide type.


The effect of pre-incubation and direct procedure for the alanine transaminase assay in two kits compared.

The kinetic measurement of serum alanine transaminase was carried out on two enzyme kits (Roche and Calbiochem), done as a direct method and a pre-incubation method. Variable results from commercial control sera and patient sera by these four procedures were obtained. Pre-incubation enzyme values were generally lower than the direct values (mean difference 10%), but there were marked differences between the results from the two kits. This finding has important significance especially for laboratories participating in external QC Programmes. Frequent precision checks of the method and a local reference range are therefore necessary.


Serum calcium ion activity. Some aspects on methodological differences and intraindividual variation.

Sera were analyzed for calcium ion activity on the Orion 99-20 and SS-20 systems. Samples from 84 healthy subjects were analyzed on both systems and, because of a change of electrode composition, 74 of them were reanalyzed after this change. Additional 89 were analyzed on only SS-20. The distribution of serum calcium ion activity was close to Gaussian. The difference between the 99-20 and SS-20 readings was 0.048+/-0.017 mmol/liter (range -0.01 to 0.08) before the electrode change and -0.085+/-0.027 mmol/liter (range -0.14 to 0.02) after. The reference ranges were 1.02--1.23 mmol/liter for 99-20 and 0.93--1.20 mmol/liter for SS-20 before the electrode change. The following characteristics may contribute to differences in the readings: higher temperature, higher flow rate, new calcium and reference electrodes, new liquid junction construction and a new pumping procedure. For 74 subjects the determination of serum calcium ion activity (99-20 system) was performed with a five year interval and the difference between the first and second measurements was -0.0046+/-0.0168 mmol/liter (mean +/-SD), ranging from -0.04 to 0.03 mmol/liter. This corresponded to an intraindividual variation of 1.5%. The corresponding variations for serum total calcium and corrected calcium were 4.8% and 3.3% respectively.


Further studies on the electrophoretic pattern of albumin in diabetic sera.

Human sera from normal subjects and diabetic patients were electrophoresed in an urea-containing gel; polyacrylamide gel and cellulose acetate membrane. In polyacrylamide gel, normal serum exhibited the fast migrating Band 2 and the slower migrating albumin Bands 4 and 5 after overnight fasting and also after glucose administration. In diabetic serum Bands 4 and 5 did not appear before or after glucose administration in low mercaptoethanol gel which abnormality resembled the pattern in C57BL/KsJ-db/db mice. In high mercaptoethanol gel, about half of the diabetic sera exhibit a delay in appearance of Bands 4 and 5, i.e., Bands 4 and 5 were not observed 30--60 min after glucose administration, which seems to resemble the pattern in C57BL/6J-ob/ob mice. Conditions of electrophoresis in urea-submerged cellulose acetate membrane (species of buffer systems, pH, ion concentration, mercaptoethanol, ethylene-diaminetetraacetic acid, Ca2+ ion, urea concentration, etc.) were observed in relation to albumin sub-separation. At pH 8.6 with barbital buffer, albumin separated into two bands, and at pH 10.6 with glycine buffer, albumin separated into four bands. Almost all diabetic sera (ca. 80%) exhibited different electrophoretic patterns from that of normal serum.


Electrolytes and norepinephrine levels in blood of patients with cystic fibrosis.

Young adults with cystic fibrosis in good to excellent condition have reduced plasma sodium and chloride and elevated plasma potassium compared to a group of healthy young adult control subjects. Blood pressure was also lower in the patients with cystic fibrosis. However, plasma norepinephrine and dopamine-beta-hydroxylase and their response to standing and isometric hand grip were normal in the cystic fibrosis patients.


Short chain fatty acids in plasma and brain: quantitative determination by gas chromatography.

A reliable and practicable method for the determination of short chain fatty acids in plasma and brain tissue is presented. The sample preparation by partition chromatography on silicic acid allows subsequently a quantitation of short chain fatty acids without interference by methylmalonic acid, or other more polar compounds. With the gas-chromatographic system 2-methylbutyrate is separated from isovalerate. Reference values are given for plasma. The system is also useful in combination with mass spectrometry.


Plasma and white blood cell ascorbic acid concentrations in patients with bronchial asthma.

Plasma cholesterol, plasma and white blood cell ascorbic acid concentrations were estimated in 30 untreated, 32 salbutamol-treated patients with bronchial asthma and in 57 normal controls. Treated and untreated asthmatics had significantly higher plasma cholesterol but lower plasma and white cell ascorbic acid levels than normal controls. A negative correlation was found between white cell ascorbic acid level and plasma cholesterol concentration in both treated and untreated asthmatics. These findings are discussed.


A highly specific 125I-radioimmunoassay for cholic acid conjugates.

Several modifications of the immunization procedure permitted development of a highly specific radioimmunoassay (RIA) for cholic acid conjugates. Antiserum was produced in guinea pigs using cholic acid-thyroglobulin complex as immunogen. 125-I-Cholyglycylhistamine was prepared as radioactive ligand according to a modification of the method of Spenney et al. (Spenney, J.G., Johnson, B.J., Hirschowitz, B.I., Mihas, A.A. and Gibson, R. (1977) Gastroenterology 72, 305--311). The association constant of the antisera to taurocholic acid was 1.8 x 10(7) l/mol, the working range of the assay between 9.5--890 pmol. Cross-reactivities of the antiserum to bile acids other than cholic acid species were less than 3%, which is lower than for any published bile acid RIA. Concentrations of cholic acid conjugates in sera obtained from 17 healthy fasting volunteers ranged from 0.4--1.9/mumul/l.


Urinary dihydroxanthopterin in the diagnosis of malignant hyperphenylalaninemia and phenylketonuria.

Assessment of urinary dihydroxanthopterin is proposed as a simple method of recognition of patients with malignant hyperphenylalaninemia (MHPA). High levels of urinary dihydroxanthopterin are found in untreated patients with phenylketonuria (PKU) or with dihydropteridine reductase (DHPR) deficiency. After dietary control of the serum phenylalanine level in PKU, the urinary dihydroxanthopterin falls to near normal levels. In DHPR deficiency urinary dihydroxanthopterin levels are high even when serum phenylalanine levels are in the range achieved on dietary treatment. Low levels would be expected in patients with defects in tetrahydrobiopterin synthesis even before dietary treatment. Confirmation of the diagnosis of different forms of MHPA then requires more detailed studies, but dietary treatment of other PKU patients can proceed with confidence.


A rapid double antibody non-equilibrium serum thyrotropin radioimmunnoassay suitable for primary neonatal hypothyroidism screening.

An improved double antibody radioimmunoassay for measuring human thyrotropin (h-TSH) in serum, suitable for neonatal primary hypothyroidism screening is described. The assay employs a non-equilibrium double antibody technique performed at an initial 37 degrees C followed by incubation at room temperature. Studies on the effects of various reaction parameters including time, temperature, antigen and antibody concentrations and the effect of various carrier proteins on the performance of standard curves were also performed. Using a 100-microliter serum sample for analysis, the assay sensitivity is routinely between 3 and 6 microU/mol of h-TSH (MRC 68/38) with interassay and intra-assay coefficient of variation values ranging from 6 to 14% over a wide range of control values. The incubation and phase separation is completed after 26 h with data available following automated counting and computation. Such a rapid assay facilitates a neonatal screening program for the early detection and treatment of primary neonatal hypothyroidism using either cord blood serum or early follow-up serum sampling in infants suspected on initial screening tests of an abnormality.


A simple specific method for the determination of the hemoglobin content of tissue homogenates.

A simple, inexpensive and specific method for the determination of hemoglobin in tissue homogenates is described. It is based on the specific binding of hemoglobin by haptoglobin and the stabilizing effect of this binding on the peroxidase activity of hemoglobin. The haptoglobin is added as serum and no purification is needed. The method gives reliable estimates of hemoglobin contents above 10 mg/l.


An improved method for the differential assay of 3-O-methylated catecholamines in human urine using ion-pair extraction and gas chromatography electron capture detection.

A gas chromatographic method is described for the quantitative determination of urinary normetadrenaline, metadrenaline and 3-methoxytyramine using electron capture detection. The N,O-dipentafluoropropionyl beta-O-ethyl derivatives of normetadrenaline and metadrenaline and the N,O-dipentafluoropropionyl derivative of 3-methoxytyramine were prepared. The amines were purified by ion exchange chromatography and ion pair extraction. Amine derivatives from urine extracts were identified definitively by mass fragmentography. The 24-h excretion value for normetadrenaline in human urine is 167 +/- 95 micrograms (mean +/- S.D.), for metadrenaline is 116 +/- 74 micrograms (mean +/- S.D.) and for 3-methoxytyramine is 82 +/- 68 micrograms (mean +/- S.D.).


A new colorimetric method for the determination of serum enzyme, gamma-glutamyl transpeptidase, cystine aminopeptidase, and leucine aminopeptidase.

A new colorimetric method for the serum enzyme assay was developed, using the color reaction of a ferrous salt complex and a primary aromatic amine. For this method new substrates containing primary aromatic amines were synthesized. The aromatic amine liberated by the enzyme reaction was measured by its color reaction with pentacyanoammineferroate treated with hydrogen peroxide. The determination was made at a wavelength of around 700 nm, at which the assay is not affected by colored substances in serum. Therefore a blank assay was not necessary. This method was applied to the assay of serum gamma-glutamyl transpeptidase, cystine aminopeptidase, and leucine aminopeptidase. The results obtained were in good agreement with those of the p-nitroanilide method also used to assay each enzyme. This new method can be used on a Technicon autoanalyzer system.


A rapid ultraviolet spectrophotometric procedure for the microdetermination of theophylline (1,3-dimethylxanthine) in plasma or serum.

An ultraviolet spectrophotometric procedure for the microdetermination of theophylline in plasma is described. Theophylline is extracted at pH 5 with chloroform. An aliquot of the solvent is shaken with molar hydrochloric acid and the absorbance of the acid layer determined at 267 nm. The resulting procedure is rapid, reliable, sensitive and virtually specific. It requires 250 microliters sample for a single estimation and has a detection threshold of less than 5 mumol/l.


Plasma spermine concentrations of patients with benign and malignant tumours of the breast or prostate.

A radioimmunoassay has been developed for the measurement of plasma spermine concentrations. The sensitivity of the method is 1 pmol spermine/100 microliters plasma and the crossreactivity was 12% with spermidine and 0.18% with putrescine. Plasma spermine levels of patients with benign and malignant tumours of the prostate or breast were measured using this technique. Concentrations were only occasionally elevated in patients with prostatic tumours compared to normal individuals and there was no difference between those men with benign (mean concn. 0.21 +/- 0.14 nmol/ml plasma) or malignant (mean concn. 0.21 +/- 0.11 nmol/ml plasma) tumours. Only 17% of the patients with breast carcinoma had elevated levels of spermine, although there was a significant difference in the concentrations of the breast cancer group of patients compared to normals. No correlation was found between elevated plasma spermine concentrations and tumour grade or presence or spread of metastases in those patients.


Plasma lipid analysis by thin-layer chromatography with flame ionisation detection and quantitation.

A method is presented whereby cholesterol, cholesterol ester, triacylglycerol, non-esterified fatty acid and polar lipid in plasma may be simultaneously quantitated. Following the addition of umbelliferyl palmitate as internal standard, plasma (0.1 ml) is extracted with chloroform/methanol (2 : 1, v/v). A portion of the extract is chromatographed on silica gel fused to a narrow cylindrical quartz rod. After chromatography the rod is traversed through a flame ionisation detector and the responses recorded and integrated. The procedure is shown to be sensitive, linear, reproducible and to compare well with conventional methods for the determination of plasma lipid classes.


The activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase in human cerebrospinal fluid.

A method for the determination of cyclic nucleotide phosphatase (CNP) activity in cerebrospinal fluid is presented. In normal CSF the activity of CNP is very low. Comparing CSF from patients with multiple sclerosis to control CSF no significant difference is found, although a small proportion of MS patients has an elevated has an elevated CNP activity in their CSF. It is postulated that similar to other substances the CNP activity in CSF probably does not derive from the central nervous system.


The production of antibodies to vitamin B-12.

The conjugation of vitamin B-12 to HSA by means of a combined ethylcarbodiimide N-hydroxysuccinimide conjugation method at pH 5.5 is described. It gave a ratio of 2.7 mol vitamin B-12/mol HSA. Five rabbits were immunized with this conjugate emulsified in Freund's complete adjuvant. After one booster injection high titre antisera (1 : 450 000--1 : 1 500 00) to vitamin B-12 were collected.


The application of a simple, sensitive disc assay for sialyltransferase activity to particulate and soluble enzyme preparations and its use in measuring enzymatic activity in human plasma samples.

A simple assay for the determination of sialyltransferase activity is described. The method involves the isolation of the radioactive reaction product on cellulose paper discs washed with trichloroacetic acid, thereby greatly facilitating the handling of large numbers of assays. This procedure is both more accurate and more sensitive than that involving precipitation with protein denaturants and separation by centrifugation, and is applicable to both soluble and particulate enzyme preparations. The application of the method to the determination of sialytransferase in human plasma is detailed. Enzyme activity is elevated two-fold in both cancer patients and patients with non-malignant disease. This suggests that the diagnostic use of determinations of sialyltransferase activity may be limited by its non-specificity.


Isolation and characterization of a polyamine-peptide conjugate from human amniotic fluid.

Significant amounts of the diamine putrescine and the polyamines spermidine and spermine could be detected in human third-trimester amniotic fluid only after acid hydrolysis. This observation was interpreted to mean that these amines existed only in conjugated form in this biological fluid. Upon fractionation by ultrafiltration 90--10% of the putrescine was associated with the 1000--10 000 dalton fraction. Spermine was identified in this fraction and in a low-molecular weight fraction presumably representing acetylated derivatives. Spermidine was entirely associated with the 10 000--30 000 dalton fraction. The putrescine conjugate was purified to homogeneity by column chromatography on Biogels P10 and P6 followed by ion-exchange chromatography on DEAE-Sephadex A-25. Molecular weight by gel exclusion using peptide standards was estimated to be approx. 4600. The UV absorption spectrum of the putrescine conjugate conformed to that expected for a polypeptide. This putrescine conjugate contained 39 identified amino acids with a combined molecular weight of 4713. Putrescine was detectable by high pressure liquid chromatography only after acid hydrolysis of the conjugate. No other polyamines were detected in these hydrolyzates, nor were any polyamines demonstable in hydrolyzates of control peptides nor in pooled column washes. The identity of the putrescine determined by high pressure liquid chromatography was confirmed by a two-dimensional thin-layer chromatography method. These results establish the in vivo production of a putrescine--polypeptide conjugate in man. Such molecular species may constitute yet another metabolic pathway for polyamines or may reflect another mode of post-translational modification of polypeptide structure and function. The qualitative and quantitative analysis of polyamine conjugate in human aminotic fluid may prove to be useful in the detection of abnormalities in fetal development.


Automated radioimmunoassay of nicotine.

We have developed an automated nonequilibrium procedure for the radioimmunoassay of nicotine. The use of a unique iodinated nicotine derivative in this procedure gave a sensitivity of 10 micrograms/l for nicotine with a between-run precision of 7.4% and within-run precision of 6.0%. Nicotine levels of 60 to 67 micrograms/ml were found in subjects 15 min after smoking one standard cigarette. The technique herein reported is a very rapid, and sensitive radioimmunoassay for nicotine and facilitates the determination of nicotine in smoking subjects during the actual process of smoking.


The clinical measurement of urinary total hydroxyproline excretion.

The effect of dietary control on the day-to-day variation in total hydroxyproline excretion has been examined in two studies. In the first, a normal volunteer ate a controlled diet containing varying gelatin supplements for several weeks. In the second, the effect of removing hydroxyproline-containing foods from the diets of 8 volunteers was examined. Both studies confirm that the day-to-day variation in total hydroxyproline excretion falls when the gelatin content of the diet is decreased, whether the results are expressed as total hydroxyproline excretion rate or as the total hydroxyproline: creatinine ratio. This fall in variation takes place within 24 h of dietary control beginning and therefore longer periods of dietary restrictions to achieve optimum precision in the measurement of total hydroxyproline are unnecessary. For some analytical methods, results are better expressed as the ratio total hydroxyproline:creatinine than as the total hydroxyproline excretion rate.


Dynamics and composition of serum lipids in hyperlipoproteinemias.

The microviscosity of serum lipids in patients with hyperlipoproteinemia and normal donors was determined by monitoring the degree of fluorescence polarization of the fluorescent probe 1,6-diphenyl-1,3,5-hexatrine (DPH). Determination of serum lipids--serum total cholesterol, serum triglycerides, and serum phospholipids--revealed that an increase in the ratio of cholesterol/triglyceride + phospholipid is accompanied by an increase in the degree of fluorescence polarization and an increase in the microviscosity values. Similar results were obtained with sonicated liposomes prepared from serum lipids extracts. This correlation between dynamics and composition of serum lipids was further supported by results obtained with an artificial model system of sonicated lipids dispersions. The results have shown that high microviscosity values are characteristic in patients with high levels of serum cholesterol, and that low microviscosity values are characteristic in patients with high levels of serum triglycerides. It is suggested therefore that this technique may serve as a basis for a rapid screening test for hyperlipoproteinemias.


An improved gas-chromatographic method for the determination of sulfated and unsulfated bile acids in serum.

This study has been aimed at improving some steps in the gas-liquid chromatographic determination of sulfated bile acids. The best conditions for the enzymatic hydrolysis with cholylglycine hydrolase of sulfolithocholylglycine and sulfolithocholyltaurine are described. Recoveries of more than 85% were obtained after prolonging the incubation to 12 h. A single-step procedure for solvolysis and methylation of bile acids was achieved by using, as a water scavenger, 2,2-dimethoxypropane added directly to the hydrolysis mixture. This procedure avoided the difficulty of the extraction of sulfated bile acids from aqueous solutions.


The radioimmunoassay of placental protein 5 and circulating levels in maternal blood in the third trimester of normal pregnancy.

A radioimmunoassay has been developed for placental protein 5 (PP5), a product of the human placenta. Circulating concentrations of PP5 were measured in the third trimester of normal pregnancy in 400 women. Concentrations of PP5 showed a skewed distribution and rose progressively to reach a plateau in the last four weeks of pregnancy. The development of this assay will permit studies on the potential clinical application of maternal PP5 levels during normal and abnormal pregnancy.


The urinary excretion of 3-methoxy-4-hydroxyphenylglycól in acute myocardial infarction.

Urinary excretion of 3-methoxy-4-hydroxyphenylglycol (MHPG), noradrenaline, adrenaline and vanillylmandelic acid (VMA) was studied in 30 patients with acute myocardial infarction. The excretion of MHPG was higher in patients with myocardial infarction than in the control group, with no difference between uncomplicated and complicated courses of the disease. The excretion of MHPG showed a negative although not significant correlation with noradrenaline and VMA during the first days of infarction and a statistically significant positive correlation on the 5th day of the disease. The possible causes of the changes observed are discussed.


Determination of noradrenaline and adrenaline in plasma by a radioenzymatic assay using high pressure liquid chromatography for the separation of the radiochemical products.

Plasma catecholamines are determined by a radioenzymatic assay using high pressure liquid chromatography for the separation of the labelled radiochemical products. Noradrenaline and adrenaline are converted to their O-methylated analogues, normethanephrine and metanephrine, by the enzyme catechol O-methyltransferase in the presence of tritiated S-adenosyl-L-methionine. Non-radioactive carriers are added, whereupon the metanephrines are extracted in an organic phase and re-extracted in an aqueous phase. The aqueous phase is dried, the residue taken up in a phosphate solution and chromatographed. The normetanephrine and metanephrine peaks are collected, converted to vanillin and assayed for radioactivity. The detection limit for noradrenaline is 32 ng/l plasma and for adrenaline 16 ng/l plasma. Intra- and interassay variables are respectively 6.2 and 10% for noradrenaline and 5.3 and 8.8% for adrenaline. The mean supine levels of noradrenaline and adrenaline in normal subjects are 355 and 61 ng/l plasma, respectively. This highly specific procedure takes two days for one person to assay 45 blood samples and can be reduced to one day if the chromatography step is automated.


Plasma somatomedin activity in vitamin A deficient children.

A role for vitamin A in the sulphation of mucopolysaccharides has been suggested. Somatomedin, a growth hormone dependent serum factor, has also been shown to stimulate the uptake of sulphate by cartilage. Therefore studies were undertaken on vitamin A deficient children to examine the possible interrelationship between vitamin A and somatomedin activity. Plasma somatomedin activity was markedly lowered in vitamin A deficient children and plasma HGH levels were in the normal range. The data suggest that vitamin A and somatomedin may be interrelated and also that plasma somatomedin activity may not always be determined by plasma growth hormone levels.


A gas chromatographic-mass spectrometric assay for nine hydroxyphenolic acids in uremic serum.

A mass fragmentographic method for the determination of m-hydroxybenzoic acid, p-hydroxybenzoic acid, o-hydroxyphenylacetic acid, m-hydroxyphenylacetic acid, p-hydroxyphenylacetic acid, vanillic acid, p-hydroxymandelic acid, homovanillic acid and vanilmandelic acid in uremic serum is described. The hydroxyphenolic acids were extracted from one ml of serum ultrafiltrate. After trimethylsilylation, the extract was analyzed by a gas chromatograph-mass spectrometer. Concentrations of nine hydroxyphenolic acids are higher in uremic serum. In particular, p-hydroxybenzoic acid and p-hydroxyphenylacetic acid are markedly increased in uremic serum. The influence of hemodialysis on the hydroxyphenolic acids was studied by quantitating the acids before and after hemodialysis.


Turbidimetric determination of lipoprotein-X in serum.

Turbidimetric determination of the cholestasis characterizing lipoprotein-X is based on the separation from the serum investigated of interfering lipoproteins by precipitation with the appropriate concentration of heparin and zinc acetate followed by centrifugation. In the supernatant the abnormal lipoprotein-X, which is not precipitated under these conditions, was determined by measuring the turbidity after addition of calcium chloride and heparin.


Derivative electrophoretograms and their applications.

A differentiator was constructed to produce the first and second derivative curves of routine cellulose acetate membrane electrophoretograms of human serum protein. The derivative curves could reveal the presence of multicomponent in each globulin fraction. The "M" components could be detected easily and semiquantitatively, when the cellulose acetate membrane electrophoretic pattern was evaluated by the derivative curves. Utilization of the derivative curves could be a useful adjunct to the detection of an "M" component since the presence of a narrow band produces a sharper peak on the second derivative curve and more indentation on the first derivative curve.


A simple method of lysosomal hydrolase assay in a single somatic cell and its application.

A new and simple assay method for lysosomal hydrolase in a single cultured skin fibroblast is described. Using 4-methylumbelliferyl-glycosides and ordinary laboratory equipment, the activity can be measured with the highest sensitivity of 0.5 pmol of 4-methylumbelliferone released from the substrates. Two systems were compared and the methods described proved reliable for the diagnosis of several variants of beta-galactosidase deficiency.


An ultrasensitive method for the measurement of human leukocyte calcium: lymphocytes.

Studies of the transport and distribution of calcium in leukocytes have been severely hampered by the inability to measure accurately and reproducibly the concentration of calcium in small numbers of cells. We have applied a recent development in analytical chemistry, the graphite furnace atomic absorption spectrophotometer, to this problem. The calcium content of human blood lymphocytes was determined by both graphite furnace and conventional flame atomic absorption spectrophotometry. The linearity, sensitivity and detection limits of the two techniques were compared. For measurement of calcium, the graphite furnace sensitivity was 55 times higher in aqueous samples and 60 times higher in cell samples than the flame technique. The detection limit of the graphite furnace was 800 times lower in aqueous samples and 1500 times lower in cell samples. The enhanced sensitivity of this technique allowed us to prepare samples with 20 times fewer blood cells. We have employed this graphite furnace technique to measure lymphocyte calcium content and its relationship to the calcium concentration and proportion of serum in the suspending medium. In the absence of serum, the lymphocyte calcium content approximately doubled as the medium calcium concentration was increased from 1 mumol/l to 0.5 mmol/l. At medium calcium concentrations of 0.5 mmol/l and above, the lymphocyte calcium content was 1.0 mmol/l cells. In medium adjusted to 2 mmol/l calcium, the lymphocyte calcium content approximately doubled as the medium serum concentration was increased from 0 to 2%. At medium serum concentrations of 2% and above, lymphocyte calcium content was 2 mmol/l cells. The exchangeable cell calcium, measured with 45Ca in the same samples, did not increase as serum was added to the medium.


Human inosine triphosphatase: catalytic properties and population studies.

Using a reliable 2-step colorimetric assay, some catalytic properties of human erythrocyte ITPase (inosine triphosphate pyrophosphohydrolase) (EC 3.6.1.19) were determined. The enzyme hydrolyses ITP, dITP (deoxyinosine triphosphate) and XTP (xanthine triphosphate) but not other nucleotides. IDP is a competitive inhibitor. Subcellular fractionation of cultured lymphoid-line cells showed ITPase activity to be in the cytosol. ATP and GTP hydrolysing activities were not enriched in the cytosol fraction. The range of activity was investigated in erythrocytes, peripheral leukocytes, amniotic fluid cells, skin fibroblasts, long term lymphoid lines and bone marrow fibroblasts. The mean ITPase activity was found to vary in different cell types from 4.9 to 294 units per mg protein. Fractionation of red blood cells by age revealed that the enzyme decays exponentially with erythrocyte age with a half-life of 35 days. The enzyme had a wide range of specific activity in erythrocytes from 150 normal randomly selected individuals (15--722 pmol/h/g Hb). Within the low range of erythrocyte specific activities (from 15 to 200) a correlation was found between an individual's erythrocyte activity and the activity in peripheral leukocytes. This correlation was not found at higher erythrocyte ITPase activities. Two individuals were detected who had erythrocyte ITPase activities which were close to the lowest ITPase activities of all samples tested. Thus, low or absent ITPase activity is an individual characteristic which may be expressed both in erythrocytes and in other cell types.


High performance liquid chromatographic determination of serum UV profiles of normal subjects and patients with breast cancer and benign fibrocystic changes.

High performance liquid chromatography (HPLC) was used to determine the UV profiles of serum samples taken postoperatively from 22 patients with histologically documented breast cancer, 8 patients with benign breast fibrocystic changes and 10 normal subjects. The analyses were performed on coded serum samples and after they were completed, the code was broken and the results correlated with the clinical data. Only one ml of serum was required for the HPLC analysis and identification. Detection limits for the nucleosides and bases were in the 10--20 pmol range and the injection volume of the deproteinated serum was 75 mul. The UV profiles of the normal subjects were very reproducible and similar to those of the patients with benign fibrocystic changes. The profiles of some of the cancer patients were distinctly different from the two other groups, 1-methylinosine and N2-methylguanosine, which were not detected in sera from normal subjects and patients with benign fibrocystic changes, were found in 45.5% and 22.7% of the cancer patients, respectively. Patients with the metastatic disease also showed elevated levels of guanosine and uridine. Only one false positive was found in the normal population. At present, it is not clear whether this indicates a subclinical manifestation of the disease and it must await further follow-up.


N-Acetyl-beta-D-glucosaminidase levels and diabetic microangiopathy.

N-Acetyl-beta-D-glucosaminidase (NAG) activity has been measured in the serum and urine of primary and secondary diabetics and in primary diabetics with microangiopathy. NAG activity has also been measured in the tears of diabetics with ocular complications and diabetics with no ocular changes. Results have shown significantly higher levels of urinary NAG in diabetics with proteinuria (p less than 0.001) and proteinuria and retinopathy (p less than 0.001). There was no correlation between urinary NAG activity and serum creatinine (r = 0.28) or urinary NAG and the degree of proteinuria (r = 0.24). Increased urinary NAG levels were also observed in secondary diabetes associated with haemochromatosis and acromegaly. Significantly higher serum NAG levels were found in newly diagnosed diabetics (p less than 0.01) and significantly lower levels in chemical diabetics (p less than 0.01). Compared to non-diabetic controls tear NAG levels were significantly higher in the diabetic controls (p less than 0.01), in diabetics with retinopathy (p less than 0.01), and in diabetics with cataract formation (p less than 0.05). An assessment of this enzyme is made in relation to the development of diabetic microangiopathy.


Plasma trypsin in chronic pancreatitis and pancreatic adenocarcinoma.

We have used a simple and precise radioimmunoassay to measure trypsin in human plasma. Fasting plasma trypsin concentrations were extremely low in patients with chronic pancreatitis with steatorrhoea (5 +/- 2 ng/ml) when compared to healthy controls (86 +/- 7 ng/ml, p less than 0.001). In patients with chronic pancreatitis but no steatorrhoea basal plasma trypsin levels were similar to those of the normal controls (99 +/- 25 ng/ml). A small but significant postprandial rise in plasma trypsin concentrations was observed in normal subjects (mean increment 15 +/- 4%, p less than 0.005, paired t test) but was absent in patients with chronic pancreatitis with steatorrhoea. In contrast to exocrine deficient chronic pancreatitis, other malabsorptive conditions associated with steatorrhoea (active coeliac disease and acute tropical sprue) demonstrated mean fasting trypsin concentrations similar to controls. Patients with adenocarcinoma of the pancreas had basal trypsin concentrations similar to healthy subjects as did patients with adenocarcinoma of the stomach, colon, rectum, brochus, and breast. In some cases measurement of plasma trypsin may be of help in the differential diagnosis of steatorrhoea.


Immunological identity of urinary alkaline phosphatase in health and disease.

Urinary alkaline phosphatase has been measured and an investigation has been undertaken involving starch gel electrophoresis after treatment of the enzyme with antisera raised against various human tissues. Urine was obtained from patients with bone and liver disease as well as pregnant women and normal persons. The enzyme level in urine in disease and pregnancy was raised above the normal but considerable overlap occurred. After electrophoresis the most usual finding was a single zone of activity although occasionally very minor zones were also present. Antiserum to kidney had the greatest affinity for the urinary enzyme. Decreasing affinity was found with intestinal and placental antiserum. No affinity was found with liver antiserum.


Fluorimetric assay of acid lipase in human leukocytes.

A fluorimetric assay for leukocyte acid lipase, the enzyme defect in Wolman disease, is described. The method requires a smaller volume of blood and can be performed more rapidly than the present colorimetric assay for the leukocyte enzyme.


Formation of an alkaline phosphatase-immunoglobulin G complex in human sera.

An electrophoretically slow-moving band of alkaline phosphatase (ALP) isoenzyme was found in four patients with chronic liver disease and in one with ulcerative colitis. Immunoelectrophoretic studies revealed that the slow band was a complex containing alkaline phosphatase and an immunoglobulin G of the lambda class. Its molecular weight was approximately 280 000. A complex molecule consisting of one molecule of immunoglobulin G and one molecule of alkaline phosphatase was proposed. The complex was similar to the liver and bone alkaline phosphatases in functional properties. Serum containing the complex was capable of binding liver and bone alkaline phosphatase isoenzymes but not the intestine or placenta alkaline phosphatase isoenzymes from normal controls. The presence of an abnormal immunoglobulin which binds liver and bone alkaline phosphatases appears to be responsible for the development of the complex. In one case of chronic liver disease, the complex disappeared after a few months.


Radioimmunoassay for human pancreatic amylase: comparison of human serum amylase by measurement of enzymatic activity and by radioimmunoassay.

A radioimmunoassay (RIA) for human pancreatic amylase has been developed for the determination of human serum amylase content. The assay was shown to be sensitive (7 ng/ml), reproducible and specific, but human pancreatic amylase and salivary amylase could not be distinguished by the antiserum used. In normal subjects, the mean concentration of amylase determined by the RIA was found to be 122.1 ng/ml (range: 55--250 ng/ml). A good correlation was observed between the concentration of amylase and its enzymatic activity in normal subjects. In some instances with high amylase activity, however, the rise in enzymatic activity was not accompanied by increasing amount of amylase content.


Porphobilinogen oxygenase from human erythrocytes.

Porphobilinogen oxygenase was isolated from red cells of healthy persons and of patients with disturbances in porphyrin metabolism. The hemolysates were purified by DEAE-cellulose chromatography and the oxygenase was eluted with either 3 mmol/l phosphate buffer (pH 7.4) or with 10 mmol/l Tris-HCl buffer (pH 7.6). The oxygenase can also be isolated by filtration of the hemolysate through Sephadex G-100. In healthy persons a mean value of 84.1 +/- 29.7 nmol of porphobilinogen consumed in 30 min per ml of blood was obtained. In patients with hepatic porphyrias (acute intermittent porphyria and porphyria cutanea tarda) the activity of porphobilinogen oxygenase was very low. In the case with acute intermittent porphyria the activity was increased when measured after storage at 4 degrees C but never reached normal values. In the cases of porphyria cutanea tarda, oxygenase activity increased after recovery and reached normal values. In patients with erythropoietic porphyria and in anemias the activity of porphobilinogen oxygenase gave high values. The erythrocyte enzyme was found to be heterogeneous when compared with the enzyme of other sources. It was only partially succinylated and was inactivated after storage for a few days at 4 degrees C. Some preparations showed the usual allosteric kinetics (n = 1.6--2.0), although Michaelian kinetics were also often observed. The enzyme was inhibited by alpha, alpha'-dipyridyl and EDTA as well as by several metals.


Differences in lipid and lipoprotein concentrations of capillary and venous blood samples.

The concentrations of lipids and lipoproteins measured in capillary blood serum taken from young adults were significantly lower than in venous blood, although they correlated satisfactorily. These differences may reflect differences in the morphological and hemodynamic conditions existing either in large veins or in the peripheral circulatory system. It was concluded that venous and capillary blood serum cannot be used interchangeably for these estimations.


Brain specific creatine kinase isoenzyme behavior in rat serum after bile acid, sodium oleate and albumin injection.

In this investigation we have shown that severe liver disease with cerebral involvement can be followed by the presence of BB creatine kinase isoenzyme in serum. Groups of rats were injected with a bile acid mixture, oleic acid, albumin and normal saline respectively. Bile acids or/and oleic acid induced BB isoenzyme to leak from central nervous tissue and this can be measured in serum. Albumin binding prevented this leakage. The experiments support our hypothesis, based on biochemical findings in human liver failure, that detergent and surface activity properties of the above mentioned components are able to produce this specific brain isoenzyme leakage.