Static tests measure chemically the content of nutrients, their active or inactive metabolites, or other related components in tissues and urine. The choice of tissue or fluid depends on the information required (short-term or long-term status, body pool or tissue store) and on the condition of the subject.
Various confounding factors affect static biochemical tests. Some are of a general kind, such as age, sex, ethnic group, physiological and hormonal status, seasonality, elevation, and thus cannot be eliminated; others are of a technical nature and can be reduced or eliminated by standardization; and others are biological or environmental (e.g., alcohol intake, smoking habits, and use of medicines). The most relevant confounding factors are considered for each method; those that occur during infection are examined separately.
Protein Nutritional Status
Total serum protein determination is very seldom used because it is no longer considered a sensitive index of status.
Plasma proteins are albumin, transport proteins (transthyretin (TTR) involved in thyroid hormone transport and formerly called prealbumin, retinol binding protein (RBP), and transferrin (TF)) and fibronectin (FB; an apsonic glycoprotein). Serum albumin, measured by an automated dye-binding method, has a rather large body pool and a long half-life and so it is a less sensitive index of immediate nutritional status. TTR, complexed with RBP in the carriage of vitamin A, TF, and FB have a smaller pool size and a shorter half-life than serum albumin and so their concentrations can change more rapidly. Therefore, they are immediate indicators of protein status. Plasma transport proteins are usually measured on radial immunodiffusion plates or alternatively with laser nephelometry. Useful commercial kits are available. Plasma fibronectin is measured only with laser nephelometry. Albumin and transport proteins are negative acute phase reactants. Other confounding effects of protein-losing diseases, such as reduced protein synthesis diseases, conditions involving an increase in plasma volume, or hemodilution and zinc depletion, have been reported. In addition, RBP is sensitive to deficiencies of vitamin A, and TF is affected by iron status. Insulin has also been demonstrated to interfere with plasma transport protein levels.
Urinary creatinine, usually measured with a colo-rimetric method (also automated), is used as a biochemical marker of muscle mass. In fact, urinary creatinine is a nonenzymatic product of creatine and cannot be reutilized. Various assumptions are required for correct urinary creatinine determination, and various confounding effects are reported (age, diet, intensive exercise, pregnancy, injury, fever, and renal diseases with impaired creatinine clearance). In a clinical setting, the creatinine/height index is preferred, but because of some limitations, it is not very useful.
Urinary 3-methyl-histidine (3-MH) can be measured by ion exchange chromatography or high-performance liquid chromatography (HPLC). 3-MH is present in myofibrillar proteins, and during breakdown it is excreted quantitatively because it cannot be reused or oxidized. Accordingly, it is used as an indicator of muscle protein turnover.
Various confounding effects are reported (sex, age, diet, intensive exercise, stress, hormonal and cata-bolic states, etc.) and so the use of the urinary 3-MH test is considered to be rather problematic.
Insulin-like growth factor-1 (IGF-1), or somato-medin C, is a regulator of anabolic properties. It has been proposed as a sensitive indicator of protein deficiency. It is assayed in serum by a radioimmu-noassay method available also in a kit. IGF-1 can also be used as a nutritional marker in adults receiving total parenteral nutrition. Confounding effects of stress, some hormonal diseases, and obesity have been reported.
Plasma amino acid levels have been used in the past to diagnose protein-energy malnutrition. The ratio of free nonessential amino acid levels (glycine, serine, glutamine, and taurine) to the essential amino acid levels (leucine, isoleucine, valine, and methionine) was proposed for the diagnosis of kwashiorkor. In children with this disease, this ratio can be much higher than the normal value of 2. Plasma amino acids were previously assessed by paper chromatographic methods; automated ion exchange or HPLC techniques are now preferred. However, in recent years there has been much less interest in this test.
A number of measures can be used to assess deficiency of essential fatty acids. In serum cholesterol esters, fatty acids determination is related to recent intake, in erythrocyte membranes it is related to intake during the previous 2 or 3 months, and in subcutaneous fat tissue it is related to intake of fatty acids for more than 1 year. Essential fatty acids are measured by gas-liquid chromatography.
Vitamin A (retinol) status can be assessed in the liver and plasma/serum. The best method is determination in the liver, but hepatic biopsy is very invasive and unsuitable in population studies. Plasma retinol is usually measured by HPLC after separation from its carrier (RBP), but its marginal values do not always reflect status because of homeostatic control and confounding effects (e.g., protein-energy malnutrition, infection, parasitic diseases, zinc deficiency, liver disorders, and chronic alcoholism). In the case of inflammation, the degree of depression of serum retinol can be quantified by assessing the concentration of certain acute phase proteins (CRP and AGP).
Because serum retinol is closely correlated with serum RBP, the measurement of this transport protein by the immunodiffusion technique or a portable apparatus has been proposed to assess vitamin A status.
The RBP:TTR molar ratio has been introduced to detect vitamin A deficiency (VAD) in the presence of inflammation. This test was based on the observation that VAD and inflammation were independent causes of low plasma RBP, whereas plasma TTR concentration was reduced only by inflammation. Nonsatisfactory results were reported from two African population groups.
The deuterated-retinol-dilution (DRD) technique is used to indirectly assess total body vitamin A reserves. A dose of deuterium-labeled retinyl acetate is given orally. After allowing time to reach equilibration (3-20 days), deuterated and nondeuterated retinol is measured by gas chromatography-mass spectrometry. A mathematical formula is used to estimate total body stores of vitamin A. Because of a set of assumptions and technical difficulties, this method is used mostly in research projects. In inflammation, the release of RBP is inhibited, so the test is probably unreliable.
In connection with vitamin A, its provitamins and non-provitamins, the carotenoids, need some consideration. ^-Carotene and a few other carotenoids play an independent and specific role in preventing oxidation, genotoxicity, and malignancy. The serum level of carotenoids is correlated with vegetable and fruit intake. Lutein is the best indicator of green leafy vegetable consumption. Lycopene is a good measure of tomato-based product consumption. a-Carotene in industrialized countries is probably a biomarker of carrot consumption and in West Africa a good marker of red palm oil consumption. The plasma level of carotenoids is measured by the HPLC system. However, there are difficulties with peak identification and quantification. Confounding effects of diet and season, sex and age, infection, smoking, and drinking habits are reported. With an appropriate HPLC system, it is possible to measure in a single assay vitamins A and E and individual carotenoids.
Vitamin D status is generally assessed by measurement of serum 25-hydroxyvitamin D (25-OHD) and in some circumstances 1,25-dihydroxyvitamin D (1,25(OH)2D). The current test for 25-OHD and 1,25(OH)2D determination in serum is by radioim-munoassay, also available as commercial kits. The HPLC method with ultraviolet detection can be used as an alternative. Confounding effects of seasons, age, sex, drugs, and liver and renal diseases are reported.
Vitamin E status can be assessed in plasma, erythrocytes, platelets, and adipose tissue. The most common and practical measure is a-tocopherol in plasma by HPLC. Because a-tocopherol is bound to lipoproteins, it is preferred to express plasma a-tocopherol relative to serum cholesterol. The determination of a-tocopherol in adipose tissue biopsy provides information on long-term nutritional status, but this test is too invasive. Confounding effects of chronic enteropathies, protein-energy malnutrition, hemolitic anemia, cholestatic liver disease, and some drugs and heavy metals are reported.
Vitamin K status requires a multiple approach including a functional test. Plasma phylloquinone is measured by reversed-phase HPLC using postcol-umn chemical reduction followed by fluorometric detection. Determination of the serum undercar-boxylated form of prothrombin (PIVKA-II) by enzyme-linked immunosorbent assay (ELISA) and urinary 7-carboxyglutamic acid by HPLC with fluorometric detection has been proposed. Confounding effects of age, sex, season, malfunction of gastrointestinal tract, osteoporosis, liver diseases, antibiotics, and other drugs are reported.
Thiamin status can be assessed by urinary excretion and erythrocyte thiamin pyrophosphate (TPP) tests. Thiamin urinary excretion is indicative of recent dietary intake; thiamin is detected fluorome-trically after conversion to thiochrome. If 24-h urine cannot be collected, thiamin should be determined in the fasting morning urine and expressed in relation to creatinine concentration. Erythrocyte TPP is indicative of long-term nutritional status and is assessed by HPLC using fluorometric detection after precolomn derivatization to thiochrome pyrophosphate. The only limitation is TPP instability, and determination should be carried out within 2 h of blood drawing. Erythrocyte TPP levels present large interindividual variation probably as a results of confounding factors (age and sex, alcohol intake, smoking habits, physical activity, and drugs).
Riboflavin status can be assessed by urinary excretion and whole blood flavinadeninedinucleo-tide (FAD) tests. Riboflavin urinary excretion is indicative of recent dietary intake; riboflavin is measured by HPLC using fluorometric detection. As for thiamin, if fasting morning urine is collected, riboflavin value is expressed in relation to milli-moles of creatinine. Confounding effects of physical activity, bed rest, chronic alcoholism, antibiotics, and other drugs are reported. Whole blood FAD is considered a reliable indicator of long-term nutritional status and is assessed by reversed-phase HPLC using fluorometric detection. This test presents some advantages over the functional test erythrocyte glutathion reductase activation coefficient (EGR-AC).
Vitamin B6 status is generally assessed by urinary 4-pyridoxic acid (4-PA) and whole blood or plasma pyridoxal-5'-phosphate (PLP) tests. The 4-PA test is indicative of recent intake but also of a deep compartment with slow elimination rate. 4-PA is measured by reversed-phase HPLC using fluorescence detection. When the completeness of 24-h collection is impossible, 4-PA is expressed in relation to millimoles of creatinine. PLP in whole blood or plasma is considered to be an indicator of depletion of vitamin B6 reserves. In whole blood, PLP can be measured by reversed-phase HPLC using fluoro-metric detection. A HPLC system with fluorescence detector for determination of vitamin B6 vitamers and pyridoxic acid in plasma is available. Plasma PLP can also be measured by radioenzymatic assay using tyrosine decarboxylase apoenzyme, which is more sensitive than other methods of analysis. Confounding effects of age and sex, acute phase status, tissue injury, catabolic state, smoking habits, alcoholism, pregnancy, drugs, physical exercise, organic diseases, and some inborn errors of metabolism are reported.
Niacin status can be assessed by measuring the two end products N'-methylnicotinamide (N'MN) and N'-methyl-2-pyridone-5-carboxamine (2-Py) in urine by HPLC. The ratio of these two urinary products is considered to be the best index of niacin nutritional status. With a single HPLC assay, the previously mentioned two nicotinamide metabolites and N1-methyl-4-pyridone-3-carboxamide (4-Py) can be measured. The ratio (2-Py + 4-Py)/N'MN is proposed; it has a diurnal variation and decreases with cold. However, for nutritional status assessment further investigation is needed.
Folate status can be assessed by serum/plasma folate, which provides information on recent intake, and erythrocyte folate, indicative of body folate stores and long-term nutritional status. Folate is measured by radioassay kits, sometimes simultaneously with vitamin B12. Less practical, although more accurate, are microbiological assays. In a EC-Flair programme intercomparison study, it was observed that radioassay tends to overestimate serum folate and presents considerable between-kit variability; improved standardization of diagnostic kits and the provision of suitable reference material are still of paramount importance. HPLC, liquid chromatography-mass spectrometry (LC-MS), and LC-MSMS methods are now available. Confounding effects of starvation, dietary folate intake and alcohol abuse, pregnancy, smoking habits, and drugs are reported for serum folate; iron deficiency, age, and other disease states are reported for erythrocyte folate.
Vitamin B12 status can be assessed by measuring serum or plasma total cobalamins and serum holo-transcobalamin II. Serum or plasma cobalamins are determined by competitive protein-binding assay. Kits are available to measure folate simultaneously. Microbiological assays tend to give lower results. Confounding effects of age, sex, impaired absorption by some diseases or drugs, myeloproliferative disorders, worm infestations, and severe liver disease are reported. Holotranscobalamin II is the transport protein of absorbed cobalamin and has been considered as an early indicator of vitamin B12 deficiency and possibly a marker of cobalamin malabsorption. Plasma holotranscobalamina II is measured by microparticle enzyme intrinsic factor assay (together with total vitamin B12) or by indirect immunoadsorption method.
Biotin status can be assessed in whole blood by microbiological assay. Radioimmunoassay tests are also available not only for plasma but also for urine. These tests give slightly higher values than microbiological assay.
Vitamin C status can be assessed by ascorbic acid in plasma, buffy-coat, and leucocytes. Ascorbic acid in plasma is considered an index of the circulating vitamin available to tissues, in buffy-coat it is indicative of the intracellular content, and in leucocytes (particularly polymorphonuclear) it is believed to be a good indicator of tissue stores. Whole blood and erythrocyte ascorbic acid determinations are considered of lesser value than plasma for ascorbic acid status assessment. Ascorbic acid in the previously mentioned blood components is measured with a dinitrophenylhydrazine assay and with a more practical HPLC method coupled with electrochemical or amperometric detectors. Also, a HPLC with fluorometric detection method is available. Confounding effects of acute stress, infection, surgery, smoking habits, chronic alcoholism, sex, and drugs are reported. The urinary excretion of ascorbic acid is an index of recent intake; because of instability of the collected sample, the determination is limited to special cases.
Essential Mineral and Trace Element Nutritional Status
Sodium and potassium in plasma/serum have little meaning in nutritional terms; total body Na or K are measured by radioisotope dilution.
Calcium status can be assessed measuring serum or plasma ionized calcium or indirectly by measuring bone mass and bone density. Plasma ionized calcium provides information on physiological function and is measured by a calcium-selective electrode; bone calcium content is an index of body calcium stores and is measured by neutron activation analysis or dual-photon absorp-tiometry. Confounding effects of venous stasis, cardiac arrest, large volumes of citrated blood infusion, and high or low pH are reported for plasma ionized calcium.
Magnesium status can be assessed by measuring magnesium in serum, erythrocyte, leucocyte, and urine. Serum magnesium is the method most commonly used. Confounding effects of haemolysis, energetic exercise, and pregnancy are reported. Ery-throcyte magnesium is considered indicative of a long-term status. Confounding effects of age, thyroid disease, and premenstrual tension are reported. Leucocyte magnesium is considered indicative of intracellular status. Urinary magnesium is used as an indicator of magnesium deficiency after a load test. Some precautions are necessary for this test. Magnesium is measured by flame atomic absorption spectroscopy (AAS) or automated colorimetric methods. The serum/plasma free ionized magnesium determination by selective electrode has been considered a better indicator of status. Further studies are required.
Iron status is assessed in relation to three stages of development of iron-deficiency anemia. In the first stage, to evaluate the size of body iron stores, serum or plasma ferritin can be measured by radiometric methods or using ELISA. Commercial kits are available. Confounding effects of infection, liver and malignant diseases, acute leukemia, Hodgkin's disease, rheumatoid arthritis, thalasse-mia major, alcohol consumption, age, and sex are reported. In the second stage, to determine the adequacy of iron supply to the erythroid marrow, serum iron (measured by the colorimetric method, available as commercial kits; AAS is not recommended because it gives higher values), plasma or serum total iron binding capacity (TIBC; by colo-rimetric or radioactive methods available as commercial kits), erythrocyte protoporphyrin (by specific hematofluorometer), and serum transferrin receptor (by ELISA using developed monoclonal antibodies) are measured. The percentage of trans-ferrin saturation is computed as follows: serum iron/TIBC x 100. Confounding effects of infection, chronic alcoholism, folate and vitamins B6, B12, and C deficiencies, acute viral hepatitis, malignancy, shock, physical trauma, pregnancy, and altitude are reported for serum iron; infection, protein-energy malnutrition, alcoholic cirrhosis, malignancy, nephrotic syndromes, entheropathy, pregnancy, viral hepatitis, and oral contraceptive intake are reported for TIBC; and infection, lead poisoning, and porphyrin disorders are reported for erythrocyte protoporphyrin. In the third stage, as indicators of iron-deficiency anemia, hemoglobin (by spectrophotometry or automatically with an electronic counter), hematocrit or packed cell volume (by specially designed centrifuge or an electronic counter), and red cell indices (mean cell volume and mean corpuscolar hemoglobin, both by electronic counter) are measured. Confounding effects of chronic infection, deficiencies of folate and vitamin B12, chronic diseases, hemoglobinopa-thies, parasitosis, sex, altitude, and smoking habits are reported. All tests of the third stage present low sensitivity and, for the confounding factors, low specificity. The measure of serum transferrin receptor seems to be a promising technique for the evaluation of iron deficiency or toxicity because it is not influenced by infection, inflammation, and chronic diseases. The assessment of serum ferritin and transferrin receptors is considered valuable in screening iron deficiency. Because the measurement of only one variable is not sufficient for the assessment of mild iron deficiency, and also to avoid other limitations, it is recommended to combine two or more independent variables.
Zinc status can be assessed by using AAS to measure zinc in plasma or serum, leucocyte and leucocyte subsets, urine, hair, nails, and saliva. Plasma or serum zinc is the method most commonly used. Many precautions are required during sample collection to avoid the influence of time of day, proximity of meal, stress, hemolysis, and contamination. There are also many pathophysiological conditions that can negatively influence specificity and sensitivity of serum zinc (e.g., infection, stress, chronic disease, exercise, oral contraceptive use, pregnancy, hypoal-buminemia, diabetes, starvation, severe malnutrition, and other catabolic conditions). Therefore, plasma zinc levels are generally considered a poor measure of marginal zinc deficiency. Leucocyte subset zinc, particularly monocyte zinc, is considered a useful indicator of zinc deficiency, but monocyte separation is difficult and a large blood sample must be collected. Zinc in other fluids or tissues is not considered a useful or reliable indicator of zinc deficiency.
Copper status is most frequently assessed in serum or plasma by AAS, even though this measure is of low sensitivity or specificity in the general population. Levels of copper in other tissues or fluids are difficult to assess or are not considered valid indices of copper status. Confounding effects of infection, inflammation, pregnancy, leukemia, Hodgkin's disease, some anemias, myocardial infarction, malabsorption, ulcerative colitis, Wilson's disease, hepatitis, high-level physical activity, cigarette smoking, age, and sex are reported.
Selenium status is usually assessed measuring plasma or serum selenium by AAS with a Zeeman background correction and also by the fluorometric technique. Although plasma Se determination provides information on short-term Se status, the determination in whole blood or erythrocytes is indicative of long-term status. Confounding effects of some inborn errors of metabolism, congestive cardiomyopathy, age, and some physiological conditions are reported.
The determination of Se in urine presents some limitations and Se levels in hair and nails display some drawbacks.
Iodine status is generally assessed by measuring urinary iodine using the colorimetric method, which reflects iodine intake within the past few days. If 24-h urine cannot be collected, iodine excretion can be expressed per gram of creatinine but only in areas with very low inter-and intraindividual variation in urinary creatinine. In clinical settings, the measurement of uptake of radioactive iodine is used.
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