Chapter 21 Laboratory Tests for the Determination of Vitamin Status
Introduction
Assessment of Vitamin Status
See Table 21-1 for laboratory tests and optimal ranges for common vitamins.1–5
TABLE 21-1 Laboratory Tests and Optimal Ranges for Common Vitamins
| NUTRIENT | TEST | ACCEPTABLE LEVEL |
|---|---|---|
| Water-Soluble | ||
| Ascorbic acid | Serum Leukocyte Load test | >0.3 mg/dL 30 mcg/108 WBCs 0.3-2.0 mg/h in control 24-49 mg/h after 500 mg |
| Biotin | 3-hydroxyisovalerate | <20 mcg/mg creatinine (overnight urine) |
| Folate | Erythrocyte folate | >160-650 ng/mL (~350 nmol/L) |
| Serum homocysteine | <10 mcmol/L | |
| Niacin | Urinary N-methylnicotinamide 2-pyridone 5-carboxamide (2-PYR) | >1.6 mg/g creatinine >1.6 mg/g creatinine |
| RBC NAD/NADP | >1.0 | |
| Pantothenic acid | Urinary pantothenic acid | >1 mg/day |
| Pyridoxine | Serum level | >50 ng/mL |
| Tryptophan load | <35 mg/24 h xanthurenic acid | |
| AST | <1.5 (ratio) | |
| ALT | <1.25 | |
| Plasma pyridoxal 5-phosphate | >30 nmol/L | |
| Urinary 4-PASerum homocysteine | >3.0 mol/d<10 µmol/L | |
| Riboflavin | EGRAC | <1.3 |
| Thiamine | RBC transketolase Whole blood thiamine (HPLC) | <15% increas e>16ng/mL |
| Vitamin B12 | Serum B12 Urinary methylmalonic acid Serum merhylmalonic acid Serum homocysteine Holotranscobalamin | >150 pg/mL <5 mcg/mg creatinine <0.45 mcmol/L <0.10 mcmol/L >30 pmol/L |
| Fat-Soluble | ||
| Vitamin A | Plasma retinol: | 15-60 mcg/dL: |
| 0-5 mo | >20 | |
| 6 mo-17 yr | >30 | |
| Adult | >20 | |
| Vitamin D | 25 (OH) vitamin D | 40-80 ng/mL |
| Vitamin E | Plasma α-tocopherol | >16.2 mcmol/L |
| α-tocopherol:cholestrol | >5.2 mcmol/L | |
| Vitamin K | % serum uncarboxylated osteocalcin | <20 ? (optimal not yet determined) |
ALT, alanine aminotranferase; AST, aspartate aminotransferase; EGOT, erythrocyte glutamic oxaloacetic transaminase; EGPT, erythrocyte glutamic pyruvic transaminase: FAD, flavin adenine dinucleotide; H2O2, hydrogen perioxide; NAD, nicotinamide adenine dinucleotide; NADP, nicotinamide adenine dinucleotide phosphate; RBC, red blood cell; WBC white blood cell.
Data from references 1-5.
Water-Soluble Vitamins
Ascorbic Acid (Vitamin C)
Assessment of vitamin C is particularly difficult because ascorbate readily oxidizes in assay samples. In addition, serum levels reflect recent dietary uptake rather than actual tissue levels. Recent research in an animal model of vitamin C deficiency (the Gulo mouse) clearly demonstrated that a dietary intake that does not lead to serum saturation of vitamin C results in tissue deficits.6 Serum saturation of vitamin C was required to achieve tissue concentrations similar to wild-type animals, which can synthesize ascorbate. In humans, maximum serum saturation from oral dosing was predicted to be roughly 1/60th of that achieved with intravenous administration, highlighting the inability of serum levels to predict optimal physiologic function.7,8 Leukocyte levels are not as susceptible to dietary intake but are also readily affected by infection, hypoglycemia, and many common prescription and over-the-counter drugs. The popular lingual ascorbate test does not appear to be reliable because it does not correlate well with leukocyte or serum levels. The loading test, if carefully controlled, is probably most accurate, although good standard ranges have yet to be determined. Finally, discovery of ascorbate-dependent enzymes involved in cell signaling pathways and epigenetic modulation offer the possibility for more functional analysis in the future, although unfortunately no definitive analysis is currently available.9
Biotin
Biotin is a vitamin B complex especially affected by oral antibiotics. Food is a poor source of this vitamin, making humans more dependent on gut flora sources. Elevation of 3-hydroxyisovalerate (3-HIA) due to deficiency of the biotin-dependent enzyme appears a useful measure. Additionally, two recent studies in which healthy volunteers were intentionally made biotin deficient suggested that elevated urinary levels of 3-HIA-carnitine might be a sensitive indicator, even for a marginal deficiency. This offers greater accuracy and is less prone to laboratory error than the traditional urinary 3-HIA.10,11 Plasma levels of 3-HIA-carnitine may also prove to be a sensitive marker and reduce the dependency on renal function for an accurate determination, an important consideration during pregnancy.12
Folate
Serum folate is too greatly affected by recent consumption to be clinically useful. Homocysteine levels may be elevated because of deficiency of vitamins B6 and/or B12 as well as folate. Erythrocyte folate is more accurate and considered a more reliable indicator of tissue status. Evaluation of other B vitamin status, particularly B12, may be necessary to rule out a folate deficiency. Although the presence of neutrophil hypersegmentation has been used to identify folate deficiency, this also occurs with B12 and iron deficiency, making it a very nonspecific marker.13
Niacin
Although measurement of nicotinic acid in the blood is not very reliable, measurement of its metabolites provides a clinically useful function assessment. Several metabolite tests are now available, including urinary levels of 2-pyridone 5-carboxymide and n-methylnicotinamide.14,15
Pantothenic Acid
Serum pantothenic acid does not correspond well with dietary intake, although erythrocyte levels are more closely correlated.16 Measurement of urinary excretion of pantothenic acid appears reliable.
Pyridoxine
Several procedures are available for assessing vitamin B6 status. Unfortunately, substantial agreement on the best methodology has not been established, because variations in phenotypes significantly alter the results of functional and loading tests. The active form of pyridoxine (pyridoxal 5′-phosphate [P5P]) is involved in some 60 enzymes, so deficient activity of these enzymes can be measured as a functional assessment of pyridoxine. Plasma levels of P5P appear to be a better functional indicator than erythrocyte levels, at least in patients with rheumatoid arthritis.17 Plasma levels below 30 nmol/L (considered borderline deficient) have independently been associated with an increased risk for coronary artery disease, with a particularly high risk when combined with high-sensitivity C-reactive protein.18,19 Although suitable for most circumstances, plasma P5P does not appear to be reliable during pregnancy or the acute phase of myocardial infarction, and alternatives should be used. Urinary 4-pyridoxic acid is a useful marker of recent intake only, whereas erythrocyte aminotransferase (EAST) and erythrocyte alanine aminotransferase (EALT) activation by pyridoxal phosphate may be a better indicator of long-term status.20 Elevated homocysteine may also be a sign of deficiency, at least in some populations.21
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