Automated and manual determination of N1-methylnicotinamide in urine.

An automated AutoAnalyzer method and a manual adaptation of the automated method are described for determining N1-methylnicotinamide (NMN) in urine. The new methods incorporate improvements presented by Pelletier and Campbell (Anal. Biochem. 3:60, 1962) for the fluorometric determination of NMN after condensation with methyl ethyl ketone (MEK). The new methods are simpler and not limited by the usage of a single brand of MEK. Various brands of MEK into which hydrogen peroxide had been added in order to catalyze the formation of the condensation product were used and found to give comparable NMN values with different urine specimens. Results of the analysis of 21 urine specimens by the new procedures agreed well with those by the method of Carpenter and Kodicek (Biochem. J. 46: 421, 1950) for which interpretation guidelines exist. Furthermore, it was found that eluates from the chromatographic purification of thiamine in urine with Decalso retained the NMN present in the original specimens, gave complete recoveries of added NMN and were free from nearly all interfering fluorescing substances; this purification would be advantageous for determining with accuracy and precision NMN levels in the urine of deficient subjects and could be used routinely when thiamine analysis has to be performed.

The nutritional and metabolic effects of a carbohydrate-rich diet in a glycogen supercompensation training regimen.

It has been suggested that glycogen depletion followed by a protein-fat diet and a carbohydrate-rich diet improves performance. This study was designed to determine the nutritional and metabolic effects of a carbohydrate-rich diet in a glycogen supercompensation training regimen. Four male subjects participated in a 5-week protocol of which the first 3 weeks were devoted to a control period and the last 2 weeks to the experimental phase of the study. The variables measured before, during, and following the experimental phase included anthropometric and basal metabolic rate measurements, urinary and serum analysis for vitamins, SMA 12/60 blood profile and aerobic performance (VO2max). Results indicated an appreciable modification of the metabolic and nutritional profile of the subjects as a result of the diets. During the protein-fat diet there was a decrease in serum glucose and resting respiratory quotient and an increase in cholesterol, blood urea nitrogen, riboflavin, and N1-methylnicotinamide excretion. Subsequent to the carbohydrate-rich diet there was an increase in triglycerides and vitamin C, riboflavin, and thiamin excretion while there was a decrease in serum blood urea nitrogen, and N1-methylnicotinamide excretion. Aerobic performance was slightly decreased and the mean postexercise lactate levels were slightly higher after the carbohydrate-rich diet. It was hypothesized that the reduced niacin intake during the carohydrate-rich diet may hamper the aerobic oxidative pathways.

Inhibition by superoxide dismutase-mimetic copper complexes of phorbol ester-induced respiratory burst in human granulocytes.

Two superoxide dismutase-mimetic lipophilic copper complexes, Cu(II)2(indomethacin)4 [Cu(II)2(indo)4] and Cu(II)2(3,5-diisopropylsalicylate)4 [Cu(II)2(3,5-DIPS)4], were tested for their effects on the respiratory burst of intact human granulocytes and on xanthine oxidase, under conditions where superoxide and hydrogen peroxide were generated. The effect of the copper complexes on these enzyme systems (as opposed to their dismutase effect on superoxide) was determined by measuring oxygen uptake with an oxygen meter. It was found that, after a short delay, both systems were inhibited markedly by micromolar amounts of these complexes. This inhibition was prevented by treatment with EDTA or catalase if added prior to starting the reaction. Similar inhibitory effects were seen using copper sulfate. It appears that these lipophilic SOD-mimetic compounds can, in the presence of H2O2 and O2-, give rise to a species that can inhibit some component of the respiratory burst oxidase or protein kinase C in intact granulocytes and xanthine oxidase in solution. The observed decrease in O2- levels observed upon addition of these compounds is likely due to inhibition of the source and not to their SOD-mimetic properties.

Production of low glucose serum for quality control and evaluation of yeast treatment, lyophilization and storage conditions on serum constituents.

In order to provide quality control serum in the hypoglycemic range, pooled human serum was treated with yeast. Yeast destroyed about 50% of the serum glucose in about 4 1/2 hrs. The yeast-treated serum remained suitable for quality control of the most commonly analyzed clinical chemistry constituents which showed only very little change in most cases. Serum triglycerides were increased by about 40% and bilirubin decreased by about 20% during the treatment. Lyophilization of serum samples (yeast-treated or not) resulted in significant decreases of some enzymes activities. Exposure at 22 degrees C of samples lyophilized (7 days) and non-lyophilized (4 days) resulted in practically no change except for certain enzymes. No significant differences were observed in the clinical chemistry measurements including glucose one month and three months after preparation of samples lyophilized (stored in a refrigerator) and non-lyophilized (store in a freezer).

Studies with an improved white cell isolation method to assess the vitamin C status in surveys.

A method for isolating white cell which employs a disposable syringe has been modified and adapted to nutrition survey work. Methylcellulose was used as a clumping agent for erythrocytes. This method was more rapid and was easier to perform than a previously recommended metrizoic acid technique which gave similar results. Average leucocyte recovery was 62% and erythrocyte contamination was 0.75 erythrocyte per leucocyte. Recovery of added ascorbic acid to white cell pellets was better than 95% and tests of the reproducibility of the assay gave a coefficient of variation of 6% for samples in the deficient range. Extraction of vitamin C from white cells was carried out in a centrifuge tube and took only 30 sec. Vitamin C was found to be stable for several weeks after freezing either the white cell extracts in dilute metaphosphoric acid or simply the white cell pellets. The method was utilized under field conditions during a small survey of Eskimos from Arctic Bay. The significance of the white cell vitamin C content as an index of nutritional status is discussed.

[Study of psychometric properties of two new questionnaires assessing beliefs in hypochondriasis in a non-clinical population]. / Evaluation des croyances reliées à la santé. Etude des propriétés psychométriques de deux nouveaux instruments evaluant les croyances présentes dans l'hypocondrie auprés d'une population non clinique.

Most instruments focussing on hypochondriasis symptoms do not have for goal to assess beliefs specifically. Instead, these instruments are used to measure specific behaviors. To assess underlying beliefs with these kinds of instruments, you have to extract false beliefs by deduction. In cognitive therapy, it is important to target erroneous beliefs in order to change them. On the other hand, existing instruments are not really suitable to target erroneous health beliefs. Even if some questionnaires are built to assess beliefs directly, it seems that they only measure the conviction of having an illness and do not assess the general health beliefs present in excessive health worriers. However, many researchers argue that this other kind of beliefs are the ones responsible in maintaining hypochondriasis symptoms. Presently, researchers assume that erroneous beliefs can maintain worries about illness among people with hypochondriasis symptoms like false beliefs about worry maintain worries in people with General Anxiety Disorder (GAD). Even if the importance of false beliefs in the maintenance of pathological worries is now recognized, most instruments on hypochondriasis symptoms do not have for goal to assess erroneous beliefs concerning worry about health. For instance, although the questionnaire Why do people worry? (WW) shows good psychometric properties and measures beliefs related to general worries, this questionnaire is not specific enough to correctly evaluate beliefs associated to health worry. A new questionnaire has to emerge in order to assess false beliefs associated to worry about health. This manuscript presents the development and the validation of a new questionnaire: the General Health Beliefs Questionnaire (GHBQ) that assesses general health beliefs, and also presents the development and the validation of a new questionnaire assessing beliefs associated to worry about health: the Why do people Worry about Health? (WW-H) . In this study, the GHBQ's and the WW-H's psychometric qualities and the factorial structure were assessed. More precisely, this study examined the factorial structure, the temporal stability, the convergent, divergent and criteria validities of the GHBQ and the WW-H. Four hundred and twenty nine French-speaking university students (non-clinical participants) completed a battery of questionnaires at the beginning of a class. The questionnaires were: The General Health Beliefs Questionnaire (GHBQ), the Why do people Worry about Health (WW-H), the Illness Worry Scale (IWS), the Beck Depression Inventory-short form (BDI-short form) and the Beck Anxiety Inventory (BAI). A second administration took place three weeks later with the same sample to test the temporal stability of the GHBQ and the WW-H. The principal component analysis with orthogonal rotation (varimax) supports a five components solution for the GHBQ: 1) magical thinking, 2) health, 3) consequences, 4) responsibility, and 5) vulnerability. The principal component analysis with oblique rotation (direct oblimin) (d=0) found a two components solution for the WW-H: 1) utility and 2) magical thinking associated to worries about health. The internal consistency of the GHBQ and the WW-H is excellent (a=.80 and a=.90, respectively). A correlation of 0.49 was found between the GHBQ and the WW-H. The correlation between the GHBQ and the IWS who evaluates the tendency to worry (r=.50) and between the WW-H and the IWS (r=.49) showed that the convergent validity of these questionnaires is adequate. On a three weeks interval, the GHBQ (r=.70) and the WW-H (r=.71) showed a satisfying temporal stability. The means of the high worriers (80 superior percentile at the IWS) (M=38.8, ET=8.93) and the means of the moderate worriers (between the 40 and the 60 percentile at the IWS) (M=32.8, ET=8.00) on the GHBQ have been compared. A significant difference has been found between the two groups [F(1,181)=23,129, p<0,001]. Also, the means of the high worriers (M=27.3, ET=8.59) and the means of the moderate worriers (M=23.8, ET=8.56) on the WW-H haveave been compared. An ANOVA has found a significant difference between these two groups [F(1,180)=7,396, p=0,007]. These results show that general health beliefs and false beliefs associated to worry about health are more often present in high worriers than in moderate worriers. The GHBQ and the WW-H allow psychologists, physicians and psychiatrists to do a quicker and more exhaustive evaluation of general health beliefs and false beliefs associated to worry about health, in less costs. These questionnaires will improve the chances of success of the hypochondriasis' treatment by helping clinicians to detect and correct false beliefs more easily.

[Prognostic value of extraembryonic components in testicular dysgerminoma (seminoma excluded)]. / Valeur pronostique des composantes extra-embryonnaires dans les dysgerminomes testiculaires (sans les séminomes).

In 32 cases of non-seminoma germinal tumours of the testis, correlations were established between the presence in situ of beta HCG and alpha foetoprotein, as demonstrated by the PAP immuno-peroxidase technique, the results of serum radioimmunological assays and the clinical course of the disease. It appeared that beta HCG-secreting mononucleate cells were present side by side with multinucleate syncytiotrophoblastic cells and that the immunohistological technique gave "earlier" results than the radioimmunological assays. The demonstration by this technique, in stage II dysgerminomas, of a cryptocellular trophoblastic carcinoma with beta HCG-secreting mononucleate cells indicates a highly malignant germinal tumour.

[Trophoblastic cryptocarcinoma with mononucleate cells, secreting beta chorionic gonadotropic hormones in seminoma. Prognostic value]. / Cryptocarcinome trophoblastique à cellules mononucléées sécrétrices d'hormones gonadotrophiques chorioniques bêta dans les séminomes. Valeur pronostique.

In 18 cases of seminoma of the testis correlations were established between beta HCG secretion, as demonstrated by the PAP immunoperoxidase technique, serum radioimmunological assays and clinical course of the disease. Mononucleate beta HCG-producing cells were found side by side with multinucleate syncytiotrophoblastic cells. This information was obtained earlier with the immunohistochemical technique than with radioimmunological assays. The immunoperoxidase technique makes it possible to detect mononucleate beta HCG-positive cell cryptocarcinomas which, when present in stage II seminomas, constitute a very dangerous dysgerminal tumour.

Automated analysis of vitamin C in pharmaceutical products.

The determination of total vitamin C in the form of both l-ascorbic acid (AA) and dehydroascorbic acid (DHAA) present in pharmaceutical preparations has been automated. Total vitamin C (completely oxidized to DHAA) was determined by reaction with 2,4-dinitrophenylhydrazine while blanks utilized the same reagent after reducing all DHAA to AA. The automated method was applicable to a variety of multivitamin preparations including those containing iron and copper. The mean recovery of L-ascorbic acid added to 11 multivitamin preparations was 99.4% with a coefficient of variation of 2.5%. In the analysis of these products, results obtained by the automated method were essentially the same as those obtained by the original manual method. For preparations containing no copper salts, the results were also comparable to those obtainable by titration with 2,6-dichloroindophenol except in 1 product which contained some DHAA.

Glass capillary or fused-silica gas chromatography--mass spectrometry of several monosaccharides and related sugars: improved resolution.

The gas chromatographic separation of several monosaccharides and related sugars derivatized by methoxylation and trimethylsilylation reactions was optimized with glass capillary (SP-2250) and fused silica (SP-2100) columns. Individual sugars included aldoses, ketoses, polyols, acidic forms and N-acetylated amino sugars. Peaks were detected by selected ion monitoring (SIM). The fused silica column gave complete resolution of all peaks (two per hexose and one per hexitol) arising from glucose, galactose, mannose, fructose, sorbitol, mannitol and dulcitol. The resolution of these sugars with the glass capillary column was not as good, but full differentiation was possible on the basis of SIM. Because the fused silica column gave a better resolution of 33 sugars tested and was more easily installed than the glass capillary column, it was utilized for quantitative analysis. A deuterated algal sugar mixture used for quantitation by isotope dilution was found to contain glucose, galactose, mannose, xylose, arabinose, ribose and rhamnose. Full recoveries were obtained of various amounts of glucose, galactose, mannose, fructose and xylose added to human serum.

Precision of glucose measurements in control sera by isotope dilution/mass spectrometry: proposed definitive method compared with a reference method.

This improved isotope-dilution gas chromatographic/mass spectrometric (GC/MS) method, in which [13C]glucose is the internal standard, meets the requirements of a Definitive Method. In a first study with five reconstituted lyophilized sera, a nested analysis of variance of GC/MS values indicated considerable among-vial variation. The CV for 32 measurements per serum ranged from 0.5 to 0.9%. However, concentration and uncertainty values (mmol/L per gram of serum) assigned to one serum by the NBS Definitive Method (7.56 +/- 0.28) were practically identical to those obtained with the proposed method (7.57 +/- 0.20). In the second study, we used twice more [13C]glucose diluent to assay four serum pools and two lyophilized sera. The CV ranged from 0.26 to 0.5% for the serum pools and from 0.28 to 0.59% for the lyophilized sera. In comparison, results by the hexokinase/glucose-6-phosphate dehydrogenase reference method agreed within acceptable limits with those by the Definitive Method but tended to be slightly higher (up to 3%) for lyophilized serum samples or slightly lower (up to 2.5%) for serum pools.

The effects of an oral contraceptive, ascorbic acid and iron on glucose and copper metabolism in the rat.

PIP: The effects of norethynodrel-mestranol, administered alone or by gavage in a diet high in ascorbic acid and/or iron, on glucose and copper metabolism were studied in the rat. After oral administration of glucose, serum glucose levels were significantly lower in animals receiving the high ascorbic acid diet (p less than .05) and in rats receiving norethynodrel-mestranol (p less than .01) than in controls. None of the dietary regimens had any effect on pancreatic histology. Norethynodrel-mestranol significantly increased serum levels of copper (p less than .05) and ceruloplasmin (p less than .01), and significantly reduced liver copper storage (p less than .01). 250 mg/kg diet of iron significantly increased copper and ceruloplasmin levels over those induced by 25 mg/kg diet of iron (p less than .05). The combination of norethynodrel-mestranol with high dietary iron and ascorbic acid intake did not adversely affect glucose tolerance and pancreatic histology.^ieng

Effects of skin preapplication treatments and postapplication cleansing agents on dermal absorption of 2,4-dichlorophenoxyacetic acid dimethylamine by Fischer 344 rats.

Various methods of preparing dermal application sites in Fischer 344 rats prior to exposure to 2,4-dichlorophenoxyacetic acid dimethylamine salt (2,4-D amine) and the effect of various cleansing agents following exposure were examined by measuring recoveries of 14C-labeled 2,4-D amine in skin, postapplication cleansing solution, blood, and urine. The middorsal area of the rat was the site of application for four treatments tested: (1) hair clipping only, (2) hair clipping followed by an epilatory cream, (3) hair clipping plus shaving with an electrical razor, and (4) as in treatment 3 followed by washing with soap and water. A last preparation was the rat's tail thoroughly brushed with soap and water. The results indicated that the tail retained greater than 75% of the material, thus preventing its absorption into the blood stream and subsequent removal by cleansing. With treatment 1 the dense short hair remaining after clipping impaired the absorption of 2,4-D as evidenced by considerably lower blood and urinary levels than treatments 2-4. With preparations 1-4, 45-61% of the dose was removed with the 7-h postapplication cleansing and a further 5-6% with the subsequent 23-h cleansing. In other studies using preparation 3 above, the following cleansing agents were tested: soap and water, water, isopropanol, acetone, and Rad-Con, a foam-producing cleanser. Rad-Con removed more 2,4-D from the skin than other cleansing agents after 7 h of exposure and more than soap and water after 23 h. The percentages of 2,4-D left on the skin following either 7- or 23-h cleansing with Rad-Con were 8-12%, nearly half those following the other cleansing agents. Cleansing agents other than Rad-Con presented little advantage over soap and water. With all cleansing agents, delaying cleansing from 7 to 23 h after exposure resulted in higher blood and urinary levels of 2,4-D measured 24 h after application.

Isotope dilution/mass spectrometry of serum cholesterol with [3,4-13C]cholesterol: proposed definitive method.

We describe a new gas-chromatographic/mass-spectrometric (GC/MS) isotope-dilution method for determination of serum cholesterol. The method has been fully optimized and documented to provide the high accuracy and precision expected for a Definitive Method. In the presence of [3,4-13C]cholesterol, cholesteryl esters in serum are hydrolyzed under optimum conditions and the entire cholesterol pool is extracted and derivatized to silyl ethers. The cholesterol derivatives are resolved from other sterols by gas-liquid chromatography on a fused silica column, and selected ions characteristic of cholesterol and the [3,4-13C]cholesterol are monitored with a GC/MS quandrupole system. We estimated the cholesterol content of samples by bracketing each sample with standards of comparable cholesterol concentration that also contained the [3,4-13C]cholesterol. The procedure was highly reproducible (CV less than 0.5%), better accuracy and precision being obtained with [3,4-13C]cholesterol than with heptadeuterated cholesterol. Mean values per gram of dry serum for one serum pool assayed by this method and that of the National Bureau of Standards differed by 0.5%. We conclude that the method satisfies the criteria for a Definitive Method.