The differential effect of angiotensin II and angiotensin 1-7 on norepinephrine, epinephrine, and dopamine concentrations in rat hypothalamus: the involvement of angiotensin receptors.

Angiotensin 1-7 has been recently claimed the active member of the angiotensins' family. In the present study we compared the effect of angiotensin II and angiotensin 1-7 on the concentration of dopamine, serotonin, epinephrine, and norepinephrine and some of their metabolites in the rat hypothalamus, where the levels of angiotensins are particularly high. Intracerebroventricular injection of angiotensin II, but not angiotensin 1-7, time-dependently elevated the levels of both epinephrine (p < 0.05) and norepinephrine (p < 0.05) in the hypothalamus and both effects could be prevented by intracerebroventricular injection of either AT(1) (candesartan), AT(2) (PD123319) or AT(1-7) (A-779) receptor antagonist. Neither angiotensin II nor angiotensin 1-7 produced any changes in the level of dopamine, dihydroxyphenylacetic acid, homovanilic acid, serotonin, 5-hydroxyindoleacetic acid, or tryptophan at any time point in comparison with the control groups. However, AT(1) but not AT(2) receptor blockade, unmasked the stimulatory effect of angiotensin 1-7 on dopamine concentration in the hypothalamus. Thus, angiotensin II and its active metabolite angiotensin 1-7 regulate selectively, albeit differentially, adrenergic, noradrenergic and dopaminergic systems in the hypothalamus, the effects that involve AT(1), AT(2) and AT(1-7) angiotensin receptors.

An automated assay for measuring serum ascorbic acid with use of 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy, free radical and o-phenylenediamine.

We developed a novel, cost-effective, and automated assay for ascorbic acid (AsA) in serum using a COBAS MIRA S analyzer (Roche Diagnostic System). Our method has a wide dynamic range and covers AsA concentrations from well below the lower reference interval to well above it. AsA is oxidized by 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy, free radical (TEMPO) to dehydroascorbic acid (DAsA). The latter condenses with o-phenylenediamine (OPDA) to form a quinoxaline derivative that absorbs light at 340 nm. The change in absorbance at 340 nm is proportional to the concentration of AsA in the specimen. The automated system permitted the assay of 65 specimens per hour at a cost of approximately US$ 0.01 per specimen for reagents. The assay can be applied directly to serum specimens (direct method) and also to sera with a prior deproteinization step with metaphosphoric acid. The detection limit for the direct serum assays is 0.8 vs. 0.4 mg/l with the deproteinization method. The recovery of AsA from a supplemented serum pool was of >95% for both procedures. We used four distinct methods on 66 patients sera. The direct method for AsA correlated well with an HPLC method (r=0.964, P<0.001); the direct method also correlated well with a method that uses AsA oxidase (r=0.975, P<0. 001). The deproteinization method correlated well with HPLC (r=0.981, P<0.001), and with the AsA oxidase procedure (r=0.994, P<0.001). Ten within-day determinations on a serum pool gave a C.V. <4.3% for both the direct and deproteinization procedures. The between-day assays of the same serum pool over 10 days gave a C.V. of <6.7% by both methods.

Threshold concentration of unbound bilirubin to induce neurological deficits in a patient with type I Crigler-Najjar syndrome.

Based on the clinical course of a 16-year-old boy with type I Crigler-Najjar syndrome, we estimated the threshold concentration of unbound bilirubin, as assayed by the horseradish peroxidase method, that apparently induces toxicity to the brain. Before the age of 15, the patient did not manifest any neurological or behavioural dysfunction despite increased bilirubin in serum. The binding affinity and the binding capacity of the patient's serum albumin for bilirubin determined when he was about 14 years old were 10(8)(mol/L)-1 and 1.01 to 1.04 mol/L, respectively. These values were nearly the same as those of normal controls reported in the literature. The total bilirubin binding capacity was greater than the patient's total bilirubin concentration, showing that his serum albumin was not saturated with bilirubin. The reserve bilirubin binding capacity (RBBC) was estimated to be 158 mumol/L and the unbound bilirubin concentration to be 15.1 nmol/L. Concentration of unbound bilirubin peaked at 21.7 nmol/L at the age of 15 years and 11 months, i.e. 2 months before the onset of difficulties in walking and speaking. At this time, the RBBC was estimated as -64 mumol/L. A peak concentration of total bilirubin, 811 mumol/L, was observed during the period of rapid loss of the ability to walk or speak. At the age of 16 years and 1 month the RBBC decreased to -98 mumol/L and the unbound bilirubin concentration to 18.8 nmol/L. Following phototherapy, the patient's neurological state returned to normal; he could speak and walk normally. At the age of 16 years and 2 months the RBBC returned to 105 mumol/L and unbound bilirubin decreased to 16.6 nmol/L. These results suggest that maintaining the concentration of unbound bilirubin at < 20 nmol/L and the total bilirubin concentration at lower than the binding capacity of serum albumin is important for prevention of neurological deficits in Crigler-Najjar syndrome. The upper limit of unbound bilirubin in such an older patient was nearly the same as that reported for newborns.

Characterization of the human gene (PTGS2) encoding prostaglandin-endoperoxide synthase 2.

The human gene (PTGS2) encoding an inducible isozyme of prostaglandin-endoperoxide synthase (prostaglandin-endoperoxide synthase 2) that is distinct from the well-characterized and constitutive isozyme (prostaglandin-endoperoxide synthase 1), was isolated using a polymerase-chain reaction-generated cDNA fragment probe for human prostaglandin-endoperoxide synthase 2. Nucleotide sequence analysis of the entire human prostaglandin-endoperoxide-synthase-2 gene demonstrated that it is more than 8.3 kb in size and consists of ten exons; this gene is very similar to the murine and chicken prostaglandin-endoperoxide-synthase-2 genes. The structures of exons in the human prostaglandin-endoperoxide-synthase-2 gene were also similar to those of the human prostaglandin-endoperoxide-synthase-1 gene (PTGS1). However, the sizes of introns in the human prostaglandin-endoperoxide-synthase-2 gene were generally smaller than those of the human prostaglandin-endoperoxide-synthase-1 gene. Primer-extension analysis indicated that the transcriptional-start site is 134 bases upstream of the translational-initiation site. The sequence of the 1.69-kb region of nucleotides preceding the transcriptional-start site and the first 0.8-kb intron contained a canonical TATA box and various transcriptional-regulatory elements (CArG box, NF-IL6, PEA-1, myb, GATA-1, xenobiotic-response element, cAMP-response element, NF-kappa B, PEA-3, Sp-1 and 12-O-tetradecanoyl-phorbol-13-acetate-response element). The nucleotide sequence of the 5'-flanking region (275 bp) of the human prostaglandin-endoperoxide-synthase-2 gene showed 63% similarity to the sequence of murine prostaglandin-endoperoxide-synthase-2/TIS10 gene, but essentially no homology to the chicken prostaglandin-endoperoxide-synthase-2 gene, and human and murine prostaglandin-endoperoxide-synthase-1 genes. A fluorescence in situ hybridization study showed that the human genes coding for prostaglandin-endoperoxide synthase 1 (PTGS1) and prostaglandin-endoperoxidase synthase 2 (PTGS2) were mapped to distinct chromosomes 9q32-q33.3 and 1q25.2-q25.3, respectively, indicating that these genes are not genetically linked.

Predeposit autologous blood transfusion in elderly patients undergoing transurethral resection of the prostate.

In order to reduce the rate of homologous blood transfusion, predeposit autologous blood transfusion has been introduced in 134 out of 266 patients undergoing transurethral resection of the prostate since February 1988. Nine (6.7%) out of 134 patients who deposited their own blood were also transfused with homologous blood. In contrast, the rate of homologous blood transfusion was 22.7% in 132 patients who did not deposit autologous blood. Overall homologous blood transfusion rate (14.7%) in the past 4.5 years has been much lower than in the previous 4 years (22.2% in 203 patients). In 6 patients in whom blood donation was performed concurrently with administration of recombinant human erythropoietin, decrease of haemoglobin level was significantly less than in those without rHuEPO. Predeposit autologous blood transfusion can reduce homologous blood transfusion rate. It is safe and easily performed, and more effective when combined with rHuEPO administration even in elderly patients undergoing TURP.

[Clinical evaluation of intra-arterial infusion of hyperthermo-chemotherapy in gastric cancer].

We performed intra-arterial infusion hyperthermochemotherapy by retaining an intra-arterial reservoir in 17 lesions of 12 patients with non-resectable, metastatic or recurrent gastric cancers. The 12 patients consisted of one with a primary gastric cancer lesion, 6 with a solitary gastric cancer lesion metastasizing to the liver, 4 with gastric cancer accompanied by hepatic metastasis, lymph node metastasis or local recurrence, and one with a gastric cancer lesion metastasizing to Douglas' pouch. A catheter was retained in the hepatic artery of all 6 patients with a solitary gastric cancer lesion metastasizing to the liver, and a catheter was retained in the aorta of the patient with a primary lesion, 3 of the 4 patients with two or more metastatic lesions, and the patient with a lesion metastasizing to Douglas' pouch. The duration of each hyperthermia session was 50 minutes, and one or two sessions were performed within a week. One course consisting of 5 or 6 sessions was repeated. Antineoplastic drugs such as MMC, 5-FU, ADR, epi-ADR, CDDP and VP-16 were injected in bolus form or administered serially through the reservoir. Nine of the 12 patients had polypharmacy. One to 3 courses or 4 to 20 sessions at maximum (average 9.8 sessions) were given. The rate of efficacy of intra-arterial infusion hyperthermochemotherapy was 44% for hepatic metastasis and 25% for lymph node metastasis. The local recurrent lesions, the lesion metastasizing to Douglas' pouch and the primary lesion did not respond to therapy.

Light has a greater effect on direct bilirubin measured by the bilirubin oxidase method than by the diazo method.

We compared the effect of light on direct-reacting bilirubin (DBIL) measurement by the bilirubin oxidase (EC 1.3.3.5; BOX) method and by the Jendrassik-Gróf diazo method. DBIL concentrations determined by the BOX method in the sera of hyperbilirubinemic infants treated with phototherapy yielded falsely higher values than those by the direct diazo method. A similar tendency was noted when DBIL concentrations in infants' sera irradiated with light in vitro were determined by both methods, although by HPLC none of these sera had detectable DBIL (i.e., conjugated plus delta bilirubin). In general, DBIL concentrations after photoirradiation remained unchanged when measured by the diazo method, but significantly increased when the BOX method was used. Indeed, photoirradiation gave rise to material that acted like a photobilirubin product, which was oxidized at pH 3.7 and therefore was measured as DBIL. Such false increases in DBIL values generated by the BOX method may have clinical diagnostic implications in monitoring jaundiced neonates and in differentiating between physiological jaundice and incipient pathological jaundice.

[Consideration on transfusion in urologic surgery--a retrospective study on homologous and autologous transfusions].

To obtain data for promoting autologous transfusion in urologic surgery, the cases of perioperative homologous transfusions in the past 5 years are reviewed. Although the mean perioperative homologous blood transfusion rate was not so high (10.2%), the rate in common urological surgery was comparatively high (eg. radical nephrectomy 39.4%, simple or palliative nephrectomy 21.6%, total nephroureterectomy 33.3%, renal allotransplantation 82.8%, total cystectomy 96.2%). Mean homologous transfusion volume in these surveys was 982 ml. Two successful cases of autologous blood transfusion by the predeposit method or intraoperative collecting method are presented. Transfusion-associated adverse effects, especially viral infection and immunosuppression, were discussed and the value of autotransfusion was stressed.

[Autotransfusion in urological surgery--experience with transurethral resection].

With the purpose to avoid the risk of homologous transfusion, autologous blood transfusion was performed in 26 patients who underwent transurethral resection of the prostate (TUR-P). Autologous blood (200-400 ml/time, 1-3 times, total 200-800 ml, mean 381 ml) was predeposited 4 days to 3 weeks prior to elected TUR-P and was transfused during or just after the operation. Since the start of this program in February 1988, homologous transfusion rate was decreased to 13.1%, whereas it has been 22.3% in 203 cases between January 1984 and January 1988. The mean hemoglobin level fell to 81.6% of the predeposition level. However, the hemoglobin level recovered to 90.1% one month after operation. The circulatory condition of the patients with autologous transfusions was stabler than that of the patients with homologous transfusion or no transfusion. No clinical hemostatic problems occurred. In cases with mild to moderate volume resection of the prostate, this autologous blood transfusion is recommended.