Conversion of vitamin B 6 compounds to active forms in the red blood cell.

In studies with pyridoxine and other B(6) compounds in blood, the active forms pyridoxal and pyridoxal phosphate were measured by differential assays using Lactobacillus casei. Red cell uptake of tritiated pyridoxine was also measured. A new metabolic pathway for conversion of pyridoxine to active forms was demonstrated in red cells. In vivo studies in normal subjects suggested that pyridoxine was taken up by red cells where it was converted to pyridoxal phosphate and then pyridoxal, followed by gradual release of a proportion of pyridoxal into plasma. In vitro incubation of pyridoxine with blood confirmed this observation. Increasing amounts of pyridoxine were taken up and converted as the amount added to blood was increased, and only very small numbers of red cells were needed to convert appreciable amounts. Conversion was markedly inhibited at temperatures lower than 37 degrees C, and stopped altogether at - 20 degrees C.Release of pyridoxal into plasma was always directly proportional to the amount of pyridoxal formed and to the volume of plasma present. That pyridoxal phosphate was not released into plasma was demonstrated in stored blood, for pyridoxine was converted mainly only as far as pyridoxal phosphate, probably due to inactivation of the phosphatase. Pyridoxal phosphate remained in the red cells. Pyridoxine was converted when incubated with washed red cells in saline or phosphate buffer suspension (0.08 M). In saline suspension, pyridoxal formed but was not released in the absence of plasma. In phosphate buffer suspension, pyridoxal phosphate was formed but was not changed to pyridoxal, probably due to inactivation of phosphatase by excess phosphate. Pyridoxamine was converted to active forms in red cells less efficiently. Pyridoxal entered red cells rapidly, equilibrating between plasma and cells within 1 min in the same ratio as pyridoxal formed inside red cells. Pyridoxal phosphate did not enter red cells in whole blood but did so readily in washed cells in saline.

Pyridoxine (pyridoxamine) phosphate oxidase activity in the red cell.

A method has been developed for the measurement of red cell pyridoxine (pyridoxamine) phosphate oxidase activity. The more stable substrate, pyridoxamine phosphate, was incubated with a red cell haemolysate and the product pyridoxal phosphate was measured by Lactobacillus casei microbiological assay. A wide range of oxidase activities was found in control subjects (6-136 ng pyridoxal phosphate/g Hb x 10(-2)). There is a close correlation between the rate of conversion of pyridoxine to pyridoxal phosphate in whole blood and the oxidase activity. There was a marked increase in both after oral riboflavin. These results suggest that the oxidase plays a large part in the regulation of vitamin B-6 metabolism in red cells.

Glutathione reductase activity and pyridoxine (pyridoxamine) phosphate oxidase activity in the red cell.

The red-cell enzymes, glutathione reductase (FAD-dependent) and pyridoxine (pyridoxamine) phosphate oxidase (FMN-dependent), were studied in control subjects. The wide range in the glutathione reductase activity correlated inversely with the percentage stimulation by FAD added in vitro, and with pyridoxine (pyridoxamine) phosphate oxidase activity. Both enzymes were stimulated after ingestion of riboflavin. The results support the suggestion that the rate of metabolism of riboflavin in the red cell controls the activity of both enzymes, and the rate of red-cell metabolism of vitamin B-6.

Rapid uptake and clearance of pyridoxine by red blood cells in vivo.

There is rapid pyridoxine (PN) uptake in vitro into red cells where it is converted to pyridoxal (PL) forms. To assess uptake in vivo, the equivalent of 48.6 and 118 mumol PN were given intravenously to a healthy female subject. Vitamin B-6 compounds were measured by a Lactobacillus casei microbiological assay in blood taken 1-60 min after injection. After either injection there was a considerable amount of PN in the red cells at 1 min but by 3 min a large amount of that PN had disappeared, mostly unaccounted for by conversion to PL forms. Although there was considerably less PN at 1 min in both red cells and plasma after the smaller injection, in the next 2 min similar amounts had left the red cells (4.59 and 4.30 mumol) and plasma (9.37 and 10.09 mumol), respectively, after the injections. Red cells, as well as plasma, may be transporting PN to other sites of metabolism in tissues.

Low red blood cell glutathione reductase and pyridoxine phosphate oxidase activities not related to dietary riboflavin: selection by malaria?

This study was designed to confirm that low dietary riboflavin does not contribute to the flavin-deficient red blood cells commonly found in subjects in Ferrara Province, northern Italy. In this area it is primarily an inherited characteristic believed to have been selected for by malaria, which was endemic from the 12th century. In parallel with assessment of daily riboflavin intake (DRI), flavin adenine dinucleotide-dependent glutathione reductase (EGR) and flavin mononucleotide-dependent pyridoxine phosphate oxidase (PPO) were measured in beta-thalassemic heterozygotes, their normal relatives, and normal spouses (representative of the normal population). In all of these groups there is a high incidence of deficiency of these flavin enzymes. We found that the majority had an adequate riboflavin intake and there was no significant correlation of EGR and PPO activities with DRI. Thus, interpretation of low EGR activity is discussed with reference to studies of EGR done to detect nutritional riboflavin deficiency in countries where there is malnutrition and endemic malaria.

Effect of light on the Lactobacillus casei icrobiological assay.

Because of the considerable differences in the reported values for serum folate in normal subjects using Lactobacillus casei as assay organism the effect of daylight when preparing media for this assay was studied. It was found that the growth properties of the medium were destroyed by direct sunlight and impaired by bright reflected sunlight, therefore reducing the sensitivity of the assay. Adequate precautions regarding exposure to light must be taken.

The measurement of serum pyridoxal by a microbiological assay using Lactobacillus casei.

A new method has been developed for the assay of serum pyridoxal using L. casei. Bound pyridoxal phosphate in serum is converted by acid hydrolysis to pyridoxal for which this organism is specific. This method proved to be considerably more sensitive than other methods so far reported in the literature. Serum pyridoxal concentrations were measured in 151 control subjects aged 17 to 80 years. The range of concentrations found was 1.5 to 13.5 ng/ml which compared well with values obtained by most workers measuring pyridoxal phosphate by enzymatic methods. A marked fall with age was confirmed, and levels in women of childbearing age were lower than in men of comparable age. Subnormal serum pyridoxal concentrations were found in 62% of patients with sideroblastic anaemia and in the majority of patients with rheumatoid arthritis, Crohn's disease, coeliac disease, and in pregnant women at term.

Demonstration of two distributions of vesicle radius in the dopamine neuron of Planorbis corneus from electrochemical data.

An electrochemical model to calculate the relative size and neurotransmitter concentration of individual nerve cell vesicles is presented to examine potentially different types of vesicles in Planorbis corneus. Amperometric current transients resulting from individual exocytosis events detected from single cells contain the information necessary to quantify vesicular neurotransmitter amount and to estimate other important cellular properties such as vesicular neurotransmitter concentration and vesicle radius. Use of a simplifying assumption that the cross-sectional area of the contents of each release event is the apparent electroactive area of the electrode and that the shape of the decreasing phase of each current transient follows Cottrell-like behavior, the Cottrell equation and Faraday's law can be combined to yield expressions for relative vesicle radius and neurotransmitter concentration. This analysis has been applied to data obtained from the cell body of the giant dopamine neuron of the pond snail P. corneus. The histogram of vesicular dopamine concentration reveals a single wide distribution and the histogram of vesicle radius reveals a bimodal radius distribution. These data strongly suggest two distinct classes of vesicle radius in the P. corneus neuron lead to the bimodal distribution of amount released reported earlier.

Calculation of transmitter concentration in individual PC12 cell vesicles with electrochemical data and a distribution of vesicle size obtained by electron microscopy.

A mathematical model is described to accurately calculate vesicle size and neurotransmitter concentration distributions from electrochemical data. This model uses parameters from electrochemical exocytosis data obtained from PC12 cells in culture to calculate a size distribution that is then correlated to the size of vesicles obtained by electron microscopy. The relative standard deviation of the size distribution calculated from electrochemical data is 25% which matches the relative standard deviation of the vesicle size distribution measured by electron microscopy. The distribution calculated from electrochemical data is normalized to the vesicle size distribution of PC12 cell vesicles obtained from electron microscopy. Calculation of a vesicular catecholamine concentration histogram from the normalized size data and electrochemical parameters is then possible for individual exocytosis events. The average vesicular catecholamine concentration for PC12 cells as calculated by this method is 148+/-7 mM. More importantly, there is a distribution of concentration rather than a constant value. Additionally, the model permits evaluation of the concentration of transmitter in each individual vesicle and vesicle size for each vesicle from electrochemical data when the overall vesicle size distribution is known.

Dopamine levels of two classes of vesicles are differentially depleted by amphetamine.

Differential depletion of neurotransmitter by amphetamine in two classes of vesicles, termed large vesicles and small vesicles, has been studied with amperometry. Carbon fiber microelectrodes have been used to monitor and quantify exocytotic events. Current transients, corresponding to individual exocytotic events, have been obtained from the cell body of the dopamine-containing neuron of Planorbis corneus. The dopamine released from individual vesicles of these cells has been compared for cells treated with D-amphetamine vs. control cells. Our results show that amphetamine has differential effects on the release of dopamine from the two classes of vesicles. Thus, it is concluded that at low concentrations, amphetamine preferentially depletes the large vesicles with a minimal effect on the small vesicles. At high concentrations, amphetamine depletes small vesicles more strongly than large vesicles although amphetamine continues to deplete the large vesicles in a dose-dependent manner. Our data appear to indicate that the two classes of vesicles observed in the Planorbis dopamine neuron might have different mechanisms associated with transmitter depletion.

Levels and subcellular localisation of pyridoxal and pyridoxal phosphate in human polymorphonuclear leukocytes and their relationship to alkaline phosphatase activity.

Neutrophil leukocytes, isolated from normal subjects and subjected to analytical subcellular fractionation by sucrose density gradient centrifugation, showed very similar cytosol distributions of pyridoxal and pyridoxal phosphate and lactate dehydrogenase. The small amounts of pyridoxal and pyridoxal phosphate associated with the dense granule fractions were not associated with the alkaline phosphatase containing granules. The levels of pyridoxal and pyridoxal phosphate were determined in neutrophils from control subjects, women in the third trimester of pregnancy and patients with chronic granulocytic leukaemia. Neutrophil pyridoxal phosphate was increased in women in the third trimester of pregnancy compared to controls, but there was little variation in the level of pyridoxal between the groups. There was no consistent correlation between the pyridoxal phosphate and the neutrophil alkaline phosphatase activity in the patient groups. Although in vitro neutrophil alkaline phosphatase rapidly hydrolyses pyridoxal phosphate, it is suggested that in vivo this is unlikely to be the principal function of the enzyme.

Chemical profiles and monitoring dynamics at an individual nerve cell in Planorbis corneus with electrochemical detection.

The identified dopamine cell of Planorbis corneus is described as a model system to study neurotransmitter storage and dynamics. Techniques developed with this model system include capillary electrophoresis with electrochemical detection and microelectrochemistry at single cells. These techniques provide a powerful combination to examine single cell neurochemistry. Whole cell and cytoplasmic dopamine concentrations have been quantified with capillary electrophoresis. Additionally, this technique has been used to profile amino acids and to quantify two compartments of neurotransmitter in a single cell. Individual exocytosis events have been monitored at the cell body of the dopamine cell of P. corneus with microelectrodes. In this case, two different types of vesicles have been identified based on the amount of transmitter released. The psychostimulant, amphetamine, has been shown to selectively affect the amount of dopamine in these vesicles with lower to higher doses affecting the larger to smaller vesicle types, respectively. Microelectrochemistry at single nerve cells has also been used to demonstrate reverse transport of dopamine across the cell membrane and to suggest a role of this process in the molecular mechanism of amphetamine.

Synchronous cancer of the colon: a case for more definitive resection in colon cancer.

Carcinoma of the colon is reported to be the most common visceral cancer today in the United States, second only to cancer of the lung as a cause of cancer-related mortality. Three consecutive cases of multiple primary colon cancers-synchronous carcinoma, two of which were additionally associated with colonic polyps, prompted a review of the literature as it related to synchronous colon cancer. The incidence of this phenomenon seems to be in the range of three percent of colon cancers. These patients tend to have a higher than usual incidence of associated polyps and of metachronous colon cancer. Inasmuch as the colons of these patients have a clear proclivity to neoplastic transformation and recurrence, subtotal colectomy as the initial curative colon procedure is practiced. This diagnosis is more often made in retrospect. It would seem that subtotal colectomy should be the initial curative procedure for any cancer of the large bowel proximal to the rectum in otherwise healthy patients less than 60 years of age.

Abdominal trauma revisited.

This article retrospectively reviews 258 consecutive abdominal trauma admissions who required laparotomy from July 1985 to June 1989 at a level one urban trauma center to define the population at risk, the patterns of injury, and the resulting morbidity and mortality. The findings were compared with a series of 252 abdominal trauma patients reported in 1974 by one of the authors. The majority of injuries in this series were gunshot wounds (GSWs) (47%), followed by stab wounds (SWs) (43%) and blunt abdominal trauma (10%). Males predominated, and 96% of patients were black. Major vascular system injury was the primary cause of death. Prompt evaluation, resuscitation, and operative intervention are mandatory in all abdominal GSW patients. More than half of those patients with major vascular system injury did not present in shock but had a high associated mortality.

Pelvic endometriosis presenting as a Supralevator abscess.

A 32-year-old woman presented with sepsis nine days after a transrectal incision and drainage of a recurrent supralevator abscess. The findings included a large mass arising from the pelvis containing multiple, leaking, and infected endometrial cysts. After a supracervical hysterectomy, bilateral salpingo-oophorectomy, sigmoid loop colostomy, appendectomy, and extensive irrigation and debridement, her condition improved with no recurrence at two-year follow-up. This case illustrates the varied presentations of endometriosis, the importance of identifying the source of a perirectal or perianal abscess, and that when a supralevator abscess develops from an intraabdominal process, the process must be addressed to prevent recurrence, fistulization, or other complications.

Mycotic aneurysms in intravenous drug abuse: diagnosis and management.

Mycotic aneurysms are caused by microorganisms in the vessel wall. The most commonly reported cause of mycotic aneurysms today is intravenous drug abuse. The diagnosis should be entertained whenever a mass is encountered over a major vessel in an intravenous drug abuser. The clinical findings often include a tender, pulsatile mass overlying an artery. The single best diagnostic test is the arteriogram. The treatment is based on early exploration with debridement of all necrotic and infected tissue, effective drainage, and appropriate antibiotic coverage.Restoration of vascular continuity is desirable when possible, and necessary when the viability of the extremity requires it. When reconstitution of the vessel is contemplated, the following principles are advocated (1) resection of all nonviable arterial wall, (2) use of monofilament synthetic suture, (3) achievement of a tension-free anastomosis with or without the use of autogenous grafts, (4) effective local drainage, and (5) appropriate antibiotic coverage.

Perforations of the gallbladder and cholecystobiliary fistulae: a review of management and a new classification.

Experiences with seven cases of gallbladder perforation managed in Kingston, Jamaica, at the District of Columbia General Hospital, and other Howard University affiliated hospitals are presented. The results of a review of 197 consecutive biliary operations at DC General Hospital for occurrence of this entity are presented.Gallbladder perforation is a complication of cholecystitis in 1 to 4 percent of cases. Niemeier(1) classified this complication in three types in 1934, and currently these are described as type 1-free perforation, type 2-perforation with abscess, and type 3-chronic perforation with cholecysto-enteric fistula.The gallbladder may, in extremely unusual occurrences, perforate into the biliary tree itself with significant operative implications, and therefore Niemeier's classification can be modified to include cholecystobiliary fistuale formation as type 4. Seven case reports manifesting all four types of perforation are presented, representing the spectrum of current treatment and diagnostic options.

Nonepithelial tumors of the stomach--gastric leiomyomatous tumors: a clinical review.

Gastric nonepithelial tumors of clinical significance are infrequently encountered. These tumors make up less than 3 percent of all gastric tumors and are predominantly composed of growths that derive from the gastric smooth muscle. The malignant nature of those leiomyomatous tumors encountered might be obvious by virtue of liver or peritoneal spread or organ invasion at the time of surgery. When this is not the case, a high frequency of mitotic figures and cell necrosis on histologic analysis, while not without exception, correlates best with malignant potential.Hemorrhage, acute or chronic, features prominently as a presenting symptom. The upper gastrointestinal series often suggests the correct diagnosis. The tumors, unlike carcinomas, exhibit little tendency to lateral mucosal and submucosal spread and to lymph node metastasis; consequently, appropriately conservative gastric resectional procedures generally suffice for treatment. Currently, surgery appears to be the only useful modality for the treatment of primary, as well as recurrent, disease.

Scrotal lymphedema praecox: disease and treatment.

Lymphedema of the scrotum and penis represents a debilitating but uncommon entity in countries spared of endemic filariasis. The clinical presentation and surgical management of one patient with idiopathic lymphedema praecox is discussed. A posterior based scrotal skin flap and a split thickness skin graft were used to reconstruct the scrotum and resurface the penis following radical excision of the edematous tissue. Limitation of the disease to the superficial tissues and relative sparing of the posterior scrotal skin represent the anatomic basis for success with this approach. The more deforming therapeutic alternative of excision of all lymphedematous tissue with transfer of the testes to the thigh and split thickness skin grafting to the exposed area would probably be best reserved for recurrence.