Prototype application of a robot in the clinical laboratory enabling fully automated quantification of fecal porphyrins.

Unpleasant specimens, sensitive analytes, and a lengthy chromatographic procedure were the main reasons we implemented fecal porphyrin analysis with a laboratory robot. We describe the system in detail and compare it with the same technique performed manually. The day-to-day variation of assays of standards was lower with the robot than with the manual operation: 8% (CV) for coproporphyrin I and 11% for protoporphyrin IX. We repeatedly analyzed a specimen from a healthy volunteer and determined that the specimen contained (in nmol/g dry wt) 7.1 (SD 0.7) for coproporphyrin I, 3.0 (SD 0.4) for coproporphyrin III, and 44.4 (SD 4.3) for protoporphyrin IX. Upper reference limits as measured in 20 healthy volunteers were 20 nmol/g dry wt for coproporphyrin I, 12 nmol/g for coproporphyrin III, and 80 nmol/g for protoporphyrin IX. We also present characteristic chromatograms for samples from various different porphyrias that exhibit abnormal fecal porphyrin excretion. Calculations of return on investment show that the robot, working at full capacity, is a profitable tool.

Aspartate aminotransferase activity in experimentally induced asymptomatic vitamin B6 deficiency in chicks.

Forty-five male Lohmann chicks were grown up to 6 weeks of age. The experimental diet containing a high protein level (30%) was aimed at increasing the metabolic need for PN. Microbiological analysis on the basal ration revealed a marginal content of 4.7 mumol PN/kg. The vitamin B6 status was assessed at the end of the experiment according to the basal activity of aspartate aminotransferase (AspAT) in plasma and in erythrocytes, and the in vitro stimulated activity with pyridoxal 5'-phosphate (PLP). None of the deficient chicks had any clinical signs attributable to malfunction of the nervous system, and they grew as well as those receiving the control diet. Vitamin B6 deficiency was biochemically confirmed by a significant depression of AspAT activity in plasma (p less than 0.001) and in erythrocytes (p less than 0.01). The addition of PLP in vitro enhanced the catalytic activity of the plasma enzyme, but had negligible effect on the erythrocyte enzyme. The degree of stimulation in vitro of the apoenzyme of AspAT not only depends on the endogenous vitamin B6 content, but also on the basal activity of the enzyme. A 15-day repletion period with a daily oral dose (50 mumol PN) did not result in a complete restoration of the enzyme activity, indicating that the availability of apoenzyme had been curtailed. This experiment demonstrated that chicks fed a high protein corn-soyamin diet with a limited amount of PN but containing Saccharomyces yeast showed no nervous signs or perosis, but significant metabolic disturbances.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma antioxidants and coronary heart disease: vitamins C and E, and selenium.

The possibility of a relation between plasma antioxidants such as vitamins C and E and selenium, and mortality from coronary heart disease (CHD) was examined. A cross-sectional survey was undertaken of random population samples of apparently healthy middle-aged men in four European regions with differing mortalities from CHD [rate/100,000 for men aged 40-49]: north Karelia (eastern Finland) (n = 99) [212/100,000]; south-west Finland (n = 85) [146/100,000]; Scotland (n = 131) [140/100,000]; and south Italy (n = 80) [43/100,000]. Median (5th-95th percentile) plasma vitamin C concentrations were lower in Scotland: 18.2 (5.7-61.3) microM than in other regions: north Karelia 28.4 (6.2-85.2); south-west Finland 33.5 (5.7-76.6); south Italy 38.0 (10.2-69.8) microM (P less than 0.001). The median levels in the four areas did not however reflect the regional CHD mortality rates. Regional differences in plasma vitamin E levels were also observed: Scottish levels were low 20.0 (12.1-29.3) microM (P less than 0.001) and did not differ between the other areas: 23.0 (16.7-35.1), 22.5 (13.7-31.6) and 23.9 (15.6-41.3) microM respectively. The vitamin E gradient could be explained in part by differences in serum cholesterol. However, cholesterol-adjusted vitamin E levels were low in the three high CHD areas: Scotland 3.41 (2.41-4.62); north Karelia 3.53 (2.67-5.18); south-west Finland 3.53 (2.58-4.92); Italy 4.81 (3.25-5.99) mumol/mmol cholesterol (P less than 0.001). Cholesterol-adjusted vitamin E was not lower in north Karelia, the higher CHD mortality area in Finland. Serum selenium values also varied with the area examined and reported low levels in Finland were confirmed. Nevertheless, selenium levels did not correlate with the reported mortality rates of CHD. Thus in our small cross-cultural study the evidence did not support our hypothesis that plasma antioxidants explain regional differences in CHD mortality.

Clinical chemical methods for the routine assessment of the vitamin status in human populations. Part III: The apoenzyme stimulation tests for vitamin B1, B2 and B6 adapted to the Cobas-Bio analyzer.

The programming of the Cobas-Bio centrifugal analyzer for kinetic tests of transketolase, glutathione reductase and aspartate amino-transferase is described. The results obtained in a population of 200 healthy people of both sexes are reported.

Assay of pyridoxal-5'-phosphate, pyridoxal and pyridoxic acid in biological material.

The two vitamin B6-vitamers having an aldehyde function are oxidised to the corresponding acids and subjected to an HPLC separation on an RP 18 phase with a solvent consisting of 5% methanol in phosphate buffer at pH 3.5. The detection is carried out by fluorometry with excitation at 318 nm and emission at 418 nm. The peaks obtained correspond to pyridoxic acid 5'-phosphate and pyridoxic acid. Pyridoxal-5'-phosphate is determined as pyridoxic acid 5'-phosphate. Pyridoxal is determined as pyridoxic acid by subtracting the amount of pyridoxic acid already existing before oxidation.

Pyridoxine status as assessed by the concentration of B6-aldehyde vitamers.

Forty-five male Lohmann chicks were randomly assigned to an adequate (30 mumol PN/kg) or a B6-deficient diet. Chicks were grown to 6 weeks of age and the vitamin B6 status was assessed according to the level of B6-aldehyde vitamers in plasma and erythrocytes. Chicks fed a limited amount of pyridoxine showed no nervous signs, but significant metabolic changes. It was found that PL was the major metabolite in plasma and only a trace of PLP was detected, suggesting a different metabolic pathway from those of other animal species or healthy humans. This particular metabolism parallels an elevated ALP concentration. The measurement of plasma PLP level routinely used for the assessment of vitamin B6 status in humans is misleading in a situation with raised ALP.

Serum levels of retinol, beta-carotene, and alpha-tocopherol in older adults.

Levels of retinol, beta-carotene, and alpha-tocopherol were determined in the serum of 415 adults over age 35 years who had volunteered in 1974 to donate blood for cancer research in Washington County, Maryland. Males had higher levels of serum retinol, lower levels of beta-carotene, and only slightly lower levels of alpha-tocopherol than did females. No clear-cut association was noted with age. Smokers had decreased serum levels of beta-carotene. Vitamin supplementation was associated with higher serum levels. Persons taking medication for high blood pressure had higher levels of retinol and alpha-tocopherol, but lower levels of beta-carotene.

Is pyridoxine an essential nutrient for bone?

Plasma PLP and PL levels were drastically reduced in the B6-deficient group. The plasma and erythrocyte activities of AspAT were depressed by 35 and 15%, respectively. The B6-deficient diet also produced a slight decrease in the plasma levels of AP (holoenzyme and bone isoenzyme) and OH-Pro. This change was accompanied by a slight increase of Pi A negative correlation was found between plasma AP and PLP.

Serum beta-carotene, vitamins A and E, selenium, and the risk of lung cancer.

We studied the relation of serum vitamin A (retinol), beta-carotene, vitamin E, and selenium to the risk of lung cancer, using serum that had been collected during a large blood-collection study performed in Washington County, Maryland, in 1974. Levels of the nutrients in serum samples from 99 persons who were subsequently found to have lung cancer (in 1975 to 1983) were compared with levels in 196 controls who were matched for age, sex, race, month of blood donation, and smoking history. A strong inverse association between serum beta-carotene and the risk of squamous-cell carcinoma of the lung was observed (relative odds, 4.30; 95 percent confidence limits, 1.38 and 13.41). Mean (+/- SD) levels of vitamin E were lower among the cases than the controls (10.5 +/- 3.2 vs. 11.9 +/- 4.90 mg per liter), when all histologic types of cancer were considered together. In addition, a linear trend in risk was found (P = 0.04), so that persons with serum levels of vitamin E in the lowest quintile had a 2.5 times higher risk of lung cancer than persons with levels in the highest quintile. These data support an association between low levels of serum vitamin E and the risk of any type of lung cancer and between low levels of serum beta-carotene and the risk of squamous-cell carcinoma of the lung.

Vitamin measurements in pooled blood samples.

From 610 blood samples collected in September 1983 in an intervention trial in Huixian, Henan Province, People's Republic of China, which were individually analyzed for retinol, beta-carotene, and alpha-tocopherol levels, 10 pools were formed from 50 individual samples, and the vitamin levels in the merged pools were also measured. Oxidation losses by repeated thawing and refreezing render the absolute levels measured in the pools smaller than the corresponding means of the individual measurements. A very good relative agreement was found between pool levels of retinol and beta-carotene and the respective means. The pooling technique could be a valuable tool in large correlation studies and has possible further applications in case-control studies.

[Long-term therapy of familial hypercholesterolemia in young patients with colestipol: availability of minerals and vitamins]. / Langzeittherapie der familiären Hypercholesterinämie bei Jugendlichen mit Colestipol: Versorgungszustand mit Mineralstoffen and Vitaminen.

Longterm treatment for 5 years of young patients with familial hypercholesterolemia was accompanied by monitoring of plasma levels of calcium, iron, sodium, parathyroid hormone and water- and fat soluble vitamins, since interference of the ion exchange resin with absorption of numerous substances as well as abnormal plasma levels of some of the above had been described in several studies. Treatment was effective in the group with good compliance (lowering of plasma cholesterol at the end of 5 years by 19% and, compared to the control group, by 23%). Adverse drug effects with respect to the above parameters were not found. Plasma levels of carotinoides and vitamin E were elevated in the patients according to elevated concentrations of lipoproteins which are carriers of these vitamins.

Serum vitamin levels and the risk of cancer of specific sites in men of Japanese ancestry in Hawaii.

Serum specimens were obtained from over 6800 men of Japanese ancestry in Hawaii from 1971 to 1975. Since then, the following numbers of newly diagnosed cancer cases have been identified: 81 colon, 74 lung, 70 stomach, 32 rectum, and 27 urinary bladder. The stored sera of the cases and 302 controls were tested to determine their beta-carotene, vitamin A, and vitamin E levels. There was no association of either vitamin A or E with any of the cancers. For serum beta-carotene, there was a significant association only with lung cancer (20.0 micrograms/dl in cases versus 29.0 in controls, P less than 0.005). The lung cancer odds ratio for men in the lowest quintile of beta-carotene was 3.4 relative to men in the highest quintile. These findings suggest that a low serum beta-carotene level is a predictor of increased lung cancer risk in men.

The influence of portosystemic shunting on zinc and vitamin A metabolism in liver cirrhosis.

To elucidate the possible role of portosystemic shunting on zinc and vitamin A deficiency which has been described in patients with cirrhosis of the liver, a study on 37 hospitalized patients with liver cirrhosis was performed. Patients with surgical portosystemic shunt were found to have a significantly lower levels of zinc, vitamin A and retinol-binding protein (RBP) than controls and patients with cirrhosis without shunt. Patients with portal hypertension--considered to have spontaneous shunting--also has lower levels than those without this symptom. A significant correlation between zinc and vitamin A and RBP levels, respectively, was found. Also an increased renal zinc output was demonstrated. An influence of portosystemic shunting on zinc deficiency and subsequent vitamin A deficiency by decreased RBP release is concluded. The importance of these metabolic disorders for clinical symptoms is discussed.

Clinical chemical methods for the routine assessment of the vitamin status in human populations. Part I: The fat-soluble vitamins A and E, and beta-carotene.

Reliable HPLC methods for the routine determination of vitamin A (only retinol, not the ester) of beta-carotene and of vitamin E in plasma are described. The results found in 150 blood donors are reported; the correlation of the concentrations of vitamin A with retinol-binding protein and of vitamin E with beta-lipoproteins are calculated.

Clinical chemical methods for the routine assessment of the vitamin status in human populations. Part II: The water-soluble vitamins B1, B2 and B6.

Simple and proven techniques for the assay of the coenzyme stimulation of the erythrocyte enzymes transketolase, glutathione reductase and glutamate oxaloacetate aminotransferase are described. The results found in a cohort of 150 blood donors and their comparison with the values for total thiamine in whole blood and pyridoxal phosphate in erythrocytes are presented.

A simple method for the determination of riboflavin in human milk.

A fluorometric method for the determination of total riboflavin in human milk is described. Flavin-adenine dinucleotide (FAD) is hydrolyzed to riboflavin by enzyme treatment. Then the fluorescence of total riboflavin is measured in the aqueous phase at pH 4.5. The coefficient of variation was found to be 2.48% (n = 58). The procedure is simple, reproducible and allows the simultaneous analysis of a considerable number of samples. The recoveries are in the range of 95.8-100.8%.

[Vitamin A concentration in plasma and ability to hear in patients with chronic alcoholic liver diseases]. / Vitamin A-Konzentration im Plasma und das Hörvermögen bei Patienten mit chronischer alkoholischer Leberschädigung.

59 patients with chronic alcoholic liver disease and with negative history of ear infection, noise exposure, head injury, use of streptomycin and without hereditary deafness underwent a basic audiologic examination. In all age groups we found a depression of the pure tone threshold on from 2,000 Hz and compared to a control group and to the norm curves (Spoor 1966). Following the Carhart-test and the acoustic facial reflex there were always signs of cochlear lesions. Fifty per cent of the alcoholics in the Carhart tone decay test showed a depression of the threshold between 10 and 30 dB. The concentration of vitamin A, RBP, beta-Carotin and zinc in the blood measured at the same time were diminished. Considering recent electron microscopic findings on the pattern of the inner ear of young rats following vitamin A deficit we suppose that the vitamin A deficit in the alcoholics leads to poor hearing.

Urinary oxalate excretion after large intakes of ascorbic acid in man.

The influence of high dose intake of ascorbic acid on the urinary excretion of oxalate was investigated in five healthy male volunteers. Oxalate was measured by a newly developed specific method using isotachophoresis. With intakes of 10 g ascorbic acid (5 X 2 g daily for 5 days; four subjects) mean urinary oxalate excretion was enhanced from about 50 mg to 87 mg (range 60 to 126 mg) per day. At least 25% of the ascorbic acid was absorbed and excreted with the urine. On discontinuing ascorbic acid administration, oxalate excretion returned to baseline values within 24 h. The time-course of oxalate excretion revealed that following the 3rd dose of 2 g ascorbic acid a plateau in urinary oxalate excretion was reached (0.6 microgram ml-1 min-1) which was not exceeded despite additional 2-g doses of ascorbic acid. On termination of ascorbic acid administration the oxalate excretion rate remained at this level for a further 6 h and then decreased to prestudy rates. No effect of high-dose ascorbic acid ingestion was found on the daily urinary excretion of creatinine, uric acid, and inorganic phosphate. Calcium excretion was slightly reduced. In comparison to the large amounts of ascorbic acid ingested, the increase in urinary oxalate excretion as measured by isotachophoresis in these healthy male volunteers was very low, and is thus similar to the change in urinary content of oxalate which results from consuming normal diets.

Absorption of large, single, oral intakes of ascorbic acid.

A male non-smoking volunteer increased his daily intake of ascorbic acid continuously by ingesting in a single, oral dose 1, 2, 3, 4 and 5 g crystalline ascorbic acid. The dose was always kept constant for 2 days and was taken with breakfast. During the regimen of ascorbic acid ingestion blood samples were taken and all urine was collected. Then, with a 5 g dose of ascorbic acid 8.17 micro Ci (170 microgram) (1-14C)ascorbic acid was ingested. Following the labelled dose radioactivity was determined in plasma, urine, and faeces. Daily ingestion of 5 g ascorbic acid was continued for 10 days. During the whole experiment the ascorbic acid concentrations in plasma and in urine were determined. The urinary excretion of unmetabolized unlabelled ascorbic acid per day was taken as index for the absorption of ascorbic acid. It decreased from 75% (1 g), 44.0% (2 g), 39%, (3g), 28% (4 g) to 20% (5 g) of the ingested ascorbic acid. The linearisation of the relationship between ingested dose and the excreted amount in urine yielded a maximum amount of 1160 mg ascorbic acid which can be absorbed in 24 h under the conditions of the experiment. The experiment with (1-14C)ascorbic acid revealed that a single oral dose of 5 g ascorbic acid is absorbed to 22.2%, in the faeces collected up to 117 h following ingestion of label only 2.8% of the radioactivity could be recovered. Several possibilities are discussed regarding the fate of the remaining unabsorbed (1-14C)ascorbic acid.

[Assay of vitamin B1 in food and biological material (author's transl]. / Zur quantitativen Vitamin-B1-Bestimmung in Nahrungsmitteln und biologischem Material.

A fluorometric method for the estimation of the vitamin B1 content in food and biological materials is described. Acid hydrolysis is followed by enzymatic treatment to liberate the vitamin B1 of the sample. Interfering substances in the extract are removed to a great extent by chromatography on Amberlite CG 50, a weakly acidic cation exchanger. After oxidation and extraction into an organic medium, the fluorescence of thiochrome is measured. The procedure is simple, reproducible and not susceptible to interference. The recoveries are good.