Detection and scavenging of hydroxyl radical via D-phenylalanine hydroxylation in human fluids.

Hydroxyl radical (.OH) is highly reactive, and therefore very short-lived. Finding new means to accurately detect .OH, and testing the ability of known .OH scavengers to neutralize them in human biological fluids would leverage our ability to more effectively counter oxidative (.OH) stress-mediated damage in human diseases. To achieve this, we pursued the evaluation of secondary products resulting from .OH attack, using a detection system based on Fenton reaction-mediated D-phenylalanine (D-Phe) hydroxylation. This reaction in turn generates o-tyrosine (o-tyr), m-tyrosine (m-tyr) and p-tyrosine (p-tyr). Here, these isomers were separated by HPLC, equipped with fluorescence detectors due to the natural fluorescence of these hydrotyrosines. By extension, we found that, adding radical scavengers competed with D-Phe on .OH attack, thus allowing to determine the .OH quenching capacity of a given compound expressed as inhibition ratio percent (IR%). Using a kinetic approach, we then tested the .OH scavenging capacity (OHSC) of well-known antioxidant molecules. In a test tube, N,N'-dimethylthiourea (DMTU) was the most efficient scavenger as compared to Trolox and N-Acethyl-L-cysteine, with NAC being the less effective. OHSC assay was then applied to biological fluid samples as seminal plasma, human serum from normal subjects and patients undergoing hemodialysis (HD), colostrum and human breast milk from mothers that received daily doses of 30g of chocolate (70% cocoa) during pregnancy. We found that a daily administration of dark chocolate during pregnancy almost doubled OHSC levels in breast milk (1.88 ± 0.12 times, p < 0.01). Furthermore, HD treatment determined a significant reduction of serum OHSC concentration (54.63 ± 2.82%, p < 0.001). Our results provide evidence that Fenton reaction-mediated D-Phe hydroxylation is a suitable method for routine and non-invasive evaluation of .OH detection and its scavenging in human biological fluids.

HPLC analysis of tetrahydrobiopterin and its pteridine derivatives using sequential electrochemical and fluorimetric detection: application to tetrahydrobiopterin autoxidation and chemical oxidation.

Tetrahydrobiopterin (BH(4)) is an essential cofactor of endothelial nitric oxide (NO) synthase and when depleted, endothelial dysfunction results with decreased production of NO. BH(4) is also an anti-oxidant being a good "scavenger" of oxidative species. NADPH oxidase, xanthine oxidase, and mitochondrial enzymes producing reactive oxygen species (ROS) can induce elevated oxidant stress and cause BH(4) oxidation and subsequent decrease in NO production and bioavailability. In order to define the process of ROS-mediated BH(4) degradation, a sensitive method for monitoring pteridine redox-state changes is required. Considering that the conventional fluorescence method is an indirect method requiring conversion of all pteridines to oxidized forms, it would be beneficial to use a rapid quantitative assay for the individual detection of BH(4) and its related pteridine metabolites. To study, in detail, the BH(4) oxidative pathways, a rapid direct sensitive HPLC assay of BH(4) and its pteridine derivatives was adapted using sequential electrochemical and fluorimetric detection. We examined BH(4) autoxidation, hydrogen peroxide- and superoxide-driven oxidation, and Fenton reaction hydroxyl radical-driven BH(4) transformation. We demonstrate that the formation of the primary two-electron oxidation product, dihydrobiopterin (BH(2)), predominates with oxygen-induced BH(4) autoxidation and superoxide-catalyzed oxidation, while the irreversible metabolites, pterin and dihydroxanthopterin (XH(2)), are largely produced during hydroxyl radical-driven BH(4) oxidation.

Isoenzyme A and urinary N-acetyl-ß-D-glucosaminidase activity in normal pregnancy.

OBJECTIVES: Urinary N-acetyl-ß-d-glucosaminidase (NAG) activity has been found to increase during normal uncomplicated pregnancy and such behavior could limit the diagnostic value of this enzyme for detection of subclinical tubular injury. The aim of this study was to evaluate urinary NAG activity and isoenzyme A in normal pregnant women at 30th week of pregnancy and in healthy women, to discriminate between physiological and lesional enzymuria. DESIGN AND METHODS: Enzyme activities in first morning fasting urine samples from 20 nonpregnant control and 20 normal pregnant women at 30th gestational week were evaluated by fluorometric methods. RESULTS: Both total and isoenzyme A activity was significantly higher ( p < 0.01) in urines of normal pregnant women compared with control urines, whereas ratio between these two parameters was significantly lower ( p < 0.001). CONCLUSIONS: The increase of urinary NAG activity during normal uncomplicated pregnancy appears to be characterized by a prevalent increase in isoenzyme A form, a finding associated with functional (not lesional) enzymuria. The fluorometric assays may represent a simple and rapid method to evaluate whether increase in urinary NAG activity represents a renal physiological adaptation during pregnancy.

The hepato-pulmonary-cutaneous syndrome: description of a case and suggestion of a unifying hypothesis.

We report a 54-year-old patient with the association of hepatic dysfunction with cyanosis, severe hypoxemia, platypnea-orthodeoxia, diffuse cutaneous spider nevi, telangiectasia, palmar erythema, digital clubbing and findings of marked intrapulmonary vascular dilation and arterovenous shunt. The diagnosis of hepato-pulmonary-cutaneous syndrome, a term we think more appropriate and inclusive than that of hepato-pulmonary syndrome for this clinicopathological picture, is proposed. The putative underlying mechanism for these connected pulmonary and extrapulmonary syndromic features is discussed.

Ruggero Oddi: 120 years after the description of the eponymous sphincter: a story to be remembered.

This article presents a short account of theories, methods, and experimental data formulated and carried out 120 years ago, by Ruggero Oddi, then a 4th-year student in medicine, about the identification of the common bile duct sphincter. A historical picture emerges which leads us to think that Oddi not only discovered the bile duct sphincter, but also described bile duct dilation after cholecystectomy and performed biliary manometry for the first time. The role of serendipity and the almost unknown contribution of Arturo Marcacci, Oddi's "maestro" are also mentioned.

Fluorimetric determination of activity and isoenzyme composition of N-acetyl-beta-D-hexosaminidase in seminal plasma of fertile men and infertile patients with secretory azoospermia.

BACKGROUND: The activity and isoenzyme composition of N-acetyl-beta-D-hexosaminidase (EC.3.2.1.52) in seminal plasma of fertile and infertile men have been evaluated. However, no data are available on the isoenzyme content in seminal plasma from patients with secretory azoospermia. METHODS: The activity and isoenzyme composition of seminal plasma from 15 normozoospermic controls and 18 patients with secretory azoospermia were determined by fluorimetric methods. 4-Methylumbelliferil-2-acetamido-2-deoxy-beta-D-glucopyranoside and 4-methylumbelliferil-2-acetamido-2-deoxy-beta-D-glucopyranoside-6-sulfate were used as fluorigenic substrates. Receiver-operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic efficiency of the assays. RESULTS: No significant difference was found in total enzyme activity between the two groups, while isoenzyme A activity was significantly lower (p=0.004) and the ratio between total enzyme activity and isoenzyme A activity was significantly higher (p=0.04) in azoospermic patients compared to controls. The diagnostic efficiency of these evaluations was low (< or =75.7%). CONCLUSIONS: Our findings show that the isoenzyme composition of N-acetyl-beta-D-hexosaminidase in seminal plasma from patients with secretory azoospermia is significantly different from controls, but this difference does not represent a useful marker of secretory azoospermia. The fluorimetric assays are simple and rapid methods for evaluating the isoenzyme composition.

Theories and controversies on urine formation.

The theories of urine formation developed in the wake of progressing scientific knowledge in renal anatomy and physiology. From the philosophical theories which for a long time swung between vitalism and mechanism, the "scientific revolution" gave a great impulse to morpho/functional unit of kidney. Bowman's secretory hypothesis, as an expression of the vitalistic based theory, describes for the first time many features of the nephron and its blood supply. New insight into the inevitable errors of Bowman led Ludwig to develop the filtration-reabsorption theory, which based its scientific approach on the emerging physics and chemistry theories. The Heidenhain's secretory hypothesis which does not admit the physical filtration in Ludwig's sense, nor the hydrostatic pressure of the blood, even though incomplete and in some part without unequivocal experimental evidence, adds a fragment to the right theory of the urine formation and heralds the modern approach to the renal function of the 20th century.

Ippolito Albertini and Michael Albertus: disparate old and innovative theories on dropsy and edema.

The concept of edema and dropsy as a part of heart and renal failure developed in the 17th and 18th centuries with the observations of Albertini, who realized that two clinical entities were derived from the blood rather than the tissues. Albertus, who lived in the same period, was the last physician to interpret fluid accumulation according to the old, scholastic and dogmatic procedures of medicine. The fundamental concepts of Albertus held little in addition to the classification and categories of the physicians of the Middle Ages. Bloody congestions were distinguished from stagnation: the former have the purpose of reducing superfluites of blood and occurred in plethoric patients. Plethora in turn is caused by the ancient villain, inculpated since Hippocrates and Galen: suppressed hemorrhoids, suppressed menstrual evacuation and cutaneous eruption driven inward. Because of its suppression, transfer of blood occurs toward the chest, which impedes thoracic expansion and contraction, then asthma and dyspnea occur. On the contrary, Albertini with his clinical and autoptic observations and pronouncements filled in the anatomical and clinical picture of fluid accumulation and created the rudiments of diagnostic criteria. Edema, dropsy, asthma, dyspnea were, according to Albertini, the signs and symptoms of heart and renal failure. Albertini was the first to point out that dyspnea is apt to arise with special rapidity when a lesion occurs in the left atrial chamber and ventricle and by implication the mitral valve. In modern physiopathological terms, he discovered the picture of pulmonary edema. To this important discovery, he added a number of extremely important comments: changes in the respiratory organs are secondary to changes in the cardiovascular system; edema that is accompanied by dyspnea also affects the viscera, most especially the lungs, and finally dropsy of the lungs must be differentiated anatomically and clinically from dropsy of the chest (hydrothorax). In other words, he depicted the anatomical and clinical picture of congestive heart failure in modern terms.

Lack of isoprene overproduction during peritoneal dialysis.

OBJECTIVE: Isoprene is the constitutive unit of isoprenoid lipids and sterols. However, it is also a potential toxic and carcinogenic agent. Recent findings of a marked and prolonged isoprene overproduction induced by hemodialysis sessions raises the question of isoprene behavior in patients on peritoneal dialysis. DESIGN: A study with repeated measures per patient and healthy control. SETTING: Nephrology and Dialysis Unit and Perugia University Medical School. PATIENTS: Sixteen consecutive patients on regular continuous ambulatory peritoneal dialysis (CAPD) were evaluated. Endogenous isoprene was analyzed using gas chromatographic assay of breath isoprene, collected at set times before and after dialysis fluid exchange. RESULTS: No significant variations were found in breath isoprene concentrations in the different samples from each patient, and levels were almost stable within the normal range of healthy controls. CONCLUSION: These results show that CAPD, unlike hemodialysis, has little or no effect on isoprene and isoprenoid-related lipid turnover. This lack of increased endogenous isoprene synthesis, in addition to being a distinctive metabolic feature of CAPD, could have important pathophysiological and clinical implications.