ABSTRACT
Methods and Their Evaluation to Estimate the Vitamin B6-Status in Human Subjects. V. S-PLP-Determination: Method and Comparison of Methods. A method to determine the plasma pyridoxal 5-phosphate (PLP) basing on the enzymatic decarboxylation of 1-14C-l-tyrosine and the measurement of the radio-active 14CO2 is described. Its efficiency is discussed in comparison with other methods. High specificity, precision, accuracy, and sensitivity are demonstrated. The developed equipment allows to analyse a great number of samples and minimizes the risk of radio-active contamination. The commercial apoenzyme preparations are different in quality and require purification. Apoenzyme lots with high genuine content of PLP are useless. Storage tests at 4 degrees C, -18 degrees C, and -196 degrees C show stability of the samples for one day, 30 days, and at least 10 weeks.
Subject(s)
Phosphoric Monoester Hydrolases/blood , Pyridoxine/blood , Carbon Radioisotopes , Decarboxylation , Humans , Tyramine/metabolism , Tyrosine Decarboxylase/metabolismABSTRACT
Some factors, which are primary independent of the vitamin B6-uptake have influence on the maximum and/or the basis activity of the EGOT and thereby finally on the alpha-value. There is a negative correlation between the age of the test persons and both the maximum and the basis activity. An insufficient supply with protein leads to lowered maximum activities, but we could not estimate a correlation between EGOT+ and protein-intake when adequate. Influences of oral contraceptives as well an enzyme induction caused by long-term high vitamin B6-uptake can not confidently be excluded. Specific differences between the enzyme activities of men and women are scarcely probable. EGOT+ and EGOT- are lowered by chronic alcohol abuse. A short-term load with alcohol does not change the results. Oral contraceptives as well as the vitamin B2 status have little if any influence on the enzyme activities. Our results show, that because of the various possible influences a single determination of the alpha-value is a doubtfull parameter to assess the vitamin B6 status of men. Only the observation of the alteration of the individual alpha-value during deficiency and substitution tests, considering at the same time some essential incidentals, can lead to well-founded statements.
Subject(s)
Aspartate Aminotransferases/blood , Pyridoxine , Adult , Age Factors , Contraceptives, Oral/adverse effects , Dietary Proteins/administration & dosage , Ethanol/pharmacology , Female , Humans , Male , Middle Aged , Nutritional Physiological Phenomena , Pyridoxine/administration & dosage , Pyridoxine/metabolismABSTRACT
A method based on the fluorimetric determination of the 4-PA-lactone is described. The applied method saves time and work compared to others. The analytical procedure may be interrupted for a few days after eluting the 4-PA from the ion exchanger as well as before measuring the transmission. Accuracy, precision and sensitivity of the method are demonstrated. Interfering substances influencing the fluorescence measurement and probable errors caused by the reference value creatinine are discussed. Storage tests applied with the urine samples at--18 degrees C have shown that 4-PA-values do not change considerably within two months.
Subject(s)
Isonicotinic Acids/urine , Pyridoxic Acid/urine , Pyridoxine/metabolism , Chromatography, Ion Exchange , Humans , Pyridoxic Acid/analogs & derivatives , Spectrometry, Fluorescence/methodsABSTRACT
Effects of physiological factors on 4-PA-excretion of more than 400 industrial workers and students were examined. Borderline values are discussed. With increasing age men as well as women showed significant higher 4-PA-values. After optimizing the vitamin B6-uptake by means of vitamin administration the differences disappear. Age-depending variations in the ability forming 4-PA are not likely. The higher 4-PA-excretion of men is probably due to better dietary supply rather than to sex differences in metabolising the vitamin. There are no considerable influences on the parameter by oral contraceptives Short-term variations of dietary vitamin B6-supply have striking effects on the 4-PA-excretion and restrict the reliability of this parameter. An insufficient vitamin B2-supply can stimulate a marginal vitamin B6-status. Alcohol consumption the day before does not change the 4-PA-excretion.
Subject(s)
Isonicotinic Acids/analysis , Pyridoxic Acid/analysis , Pyridoxine/metabolism , Adult , Age Factors , Evaluation Studies as Topic , Female , Humans , Male , Middle Aged , Reference Values , Sex FactorsABSTRACT
A semiautomatic method to determine the activation coefficient of the glutamic-oxaloacetic-transaminase in the erythrocytes is described. This method is compared with other methods and possible sources of error are discussed. Colorimetric methods show considerable disadvantages, such as lack of precision or unspecific hydrazone formation. The kinetic tests avoid these problems. This is in part due to the fact that enzyme stimulation by excessive PLP-addition as used in our procedure bears practical and theoretical advantages. Nevertheless, comparison with published data on vitamin B6 status remains difficult because of insufficient standardization of the used methods. To validate our method we performed experiments testing the stability of the samples, short-term physiological changes and influence of the chelating agent EDTA on transamination. Heparinised blood samples can be stored only for one day with changing at 4 degrees C or room temperature. Storing of the erythrocytic suspension at deep-freezing temperature (-18 degrees C) leads to considerable changes of results. When kept under liquid nitrogen there is no change of the alpha-values for at least 45 days. The addition of EDTA to the samples is known to exclude the influences of cations, such as non enzymatic transamination of a direct alteration of the enzyme activity and therefore EDTA should be added to improve standardisation of the method. The described procedure is a fast, reliable and precise way to determine the alpha-EGOT.
