Vitamin vitamin status assays

Vitamin Bg Status Is Assessed by Assaying Erythrocyte Aminotransferases... 

Microbiological assays for vitamin Bi2 are diverse most are simple, but require some experience and confidence. In our hands they have proved uniformity reliable for evaluating the vitamin Bi2 status of patients and are extremely useful research tools. Perhaps too much emphasis has been placed upon space, good and meticulously cleaned equipment, and trained personnel (G18). The Bi2 assays to be discussed here have been developed as practical methods. Four organisms have... 

Vitamin B12 status is usually determined by measuring vitamin B12 concentrations in serum by competitive protein-binding radioimmunoassay. In such an assay, the vitamin B12 in the patient s serum and the unlabeled B12 in the... 

A number of assay methods for vitamin Bu status, and for testing defects in the pathway of vitamin B,j absorption, arc listed here ... 

The assay for serum Bn levels is a direct lest, as it measures the concentration of the vitamin itself. The assay of MM A levels is a functional test of B12 status, as it measures a compound whose metabolism is dependent on the correct functioning of vitamin Bn- The results of the MMA test reflect the activity of methylmalonyl-CoA mutase in the liver It is thought that the functional test is more valuable than the direct test. Serum vitamin Bij levels may not reflect the functioning of the B j-requiring enzymes of the cell. Serum Bii levels may sometimes be withm the normal range despite an increased excretion of MMA. Normal scrum Bt2 values range from 0.2 to 1,0 ng/ml. Normal serum MMA levels range from 20 to 75 ng/ml, and normal urinary MMA levels from 0,6 to 3-0 pg/ml. 

Formation of a blood clot results in a precipitous increase in the viscosity of the blood sample. The event of dotting is initiated by adding calcium ions, to replace those chelated by the citrate, and tissue factor. Tissue factor is a lipid-rich protein that activates the blood clotting cascade. The formahon of the viscous blood clot within seconds indicates normal vitamin K status. Delayed formation of the clot indicates a deficiency. Restoration of a normal clotting time with vitamin K therapy is required to prove that the subject had been deficient, that is, that the defect in the clotting assay was the result of K deficiency, rather than some other pnO blem. 

Osteocalcin Assays as a Possible Sensitive Test for Vitamin K Status... 

The basal activity of aminotransferase fell by 50% during consumption of the Bt-deficient diet. The stimulation occurring with addition of PLP to the enzyme incubation mixtures rose in this period from 200% (twofold stimulation) to 400% (fourfold). The basal activity of the aminotransferase, and probably of ail enzymes of the body, varies from subject to subject. It may even vary with repeated enzyme assays using the same sample of red blood cells. Thus, the basal activity is not used to assess vitamin Be, status. The percentage stimulation is relatively constant in normal subjects and is thus a more useful indicator of B status. It should be noted that the storaffe of ivti blood teiis can lead to gradual release of the cofactor from the enzyme thus, an artefactual diagnosis of Bh deficiency is possible. 

Flavins are lost from the body as intael riboflavin, rather than as a breakdown product of riboflavin. Hence, vitamin status may be assessed by measuring the level of urinary riboflavin. Generally, the loss of 30 ig of riboflavin/g creatinine or less per day indicates a deficiency. This metht>d of assessment is not preferred because it is influenced by a number of factors unrelated to vitamin status. Another problem with this method is its great sensitivity to a short-term deficiency thus, it does not necessarily reflect the true concentrations of FAD and FMN in tissues. The most reliable way to assess riboflavin status is by a functional test. The test involves the assay of glutathione reductase, using red blood cells as the source of... 

Glutathione is discussed further in the section on selenium and glutathione in Chapter 10. The enzyme assay is conducted using glutathione reductase extracted from red blood cells with and without added FAD. Chmnic consumption of a diet deficient in riboflavin allows the continued synthesis of a variety of flavoproteins, but results in the accumulation of apoenzyme without its conversion to holoen-zyme. Addition of chemically pure FAD to a biological fluid containing apoenzyme results In the stimulation of enzyme activity because of the formation of the holoenzyme. It is this stimulation of enzyme activity that is used to determine vitamin status in humans. 

Thiamin status has been assessed by direct tests involving the measurement of thiamin levels in the blood or urine. The vitamin can be assayed by the thiochrome method or by microbiological assays. The disadvantage of these methods is that thiamin levels in normal individuals can vary greatly. The test organism used for microbiological assays may be Lactobacillus viridescens or Lactobacillus fermenti. 

Both direct and indirect (ftmctional) methods are available for assessing vitamin B status. The indirect tests include assays for urinary and serum concentrations of methylmalonic acid, plasma homocysteine, the deoxyuridine suppression test, and the vitamin B12 absorption test. Cyto-chemical staining of red blood cell (RBC) precursors and the test for IF blocking antibodies are also ancillary methods for assessing vitamin B12 status. 

Tanumihardjo SA, Koellner PG, Olson JA. The modified relative-dose-response assay as an indicator of vitamin A status in a population of well-nourished American children. Am J Clin Nutr 1990 52 1064-7. 

Tanumihardjo SA, Muhilal, Yuniar Y, Permaesih D, Sulaiman Z, Karyadi D, Olson JA. Vitamin A status in preschool-age Indonesian children as assessed by the modified relative-dose-response assay. Am J Clin Nutr 1990 52 1068-72. 

A Saturation of Biochemical Function. A reliable biochemical indicator is required. For niacin, which NAD" - or NADP -containing enzyme should be selected Which transaminase will be the indicator for pyridox-ine Which function of vitamin A should be j selected for retinol, vision in the rods or cell differentiation As noted from Table 8.2, many of the assays for vitamin status have significant limitations to estimate reliable doses. 

Subsequently, there is a decrease in urinary excretion of the vitamin, which in mrn is followed by diminished tissue concentrations of the vitamin. The most common measurements of vitamin status are assays of circulating amounts in plasma or serum. Assays of biochemical or metabolic function of the vitamin are more likely to reflect body stores than are serum concentrations. Most of these functional assays use erythrocyte or leukocyte extracts to determine apoenzyme activity, which is dependent on the vitamin coenzyme (see Table 135-9). 

Vitamin C status can be assessed by measuring plasma levels or urinary excretion. However, due to practical disadvantages, e.g., quantitative sampling of urine and instability of vitamin C, determination of vitamin C in urine has been mainly replaced by determination of plasma levels. Methods include direct determination by FIPLC, or automated assays based on a derivatization of ascorbic acid forming colored or fluorescent derivatives. While a plasma concentration <11.4p.molH is widely used to characterize deficiency, literature data signifying adequate status vary from >17 to 28.4p.moll. ... 

The criteria for the diagnosis of vitamin deficiency are specific for every single vitamin. However, some common rules can be applied to most of the B vitamins. First, it has to be stated that, in clinical practice, some vitamins (biotin, pantothenic acid, niacin) are hardly ever measured and therefore data on vitamin status are hardly available. Other vitamins like folate and cobalamin are measured regularly using commercial assays. 

Some investigators have used the level of RBP in serum to assess vitamin A status independent of a direct determination of the vitamin. The basis for this approach is that normally vitamin A and RBP circulate in a 1 1 molar complex with little unbound RBP or retinyl ester present. A radial immunodiffusion assay... 

---