Laboratory Tests for the Determination of Vitamin Status

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Chapter 21 Laboratory Tests for the Determination of Vitamin Status

image Assessment of Vitamin Status

See Table 21-1 for laboratory tests and optimal ranges for common vitamins.15

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

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

Fat-Soluble Vitamins

Vitamin A

Although liver biopsy is the most accurate method of vitamin A assessment, other less invasive and less expensive methodologies are more appropriate. As with most other nutrients, serum levels of vitamin A fall significantly only after tissue reserves have been depleted, and are subject to numerous laboratory challenges as well as other factors, (e.g., infection, protein status). Serum retinol binding protein is sometimes used as an alternative to serum retinol, because it avoids many of these complications, although it is also susceptible to artificial decreases due to inflammation or protein malnutrition.33 Optimal cutoffs have not been clearly established for retinol binding protein, although levels greater than 0.825 µmol/L have been suggested for children and greater than 1.05 µmol/L for adults.34,35 The dark adaptation test detects early deficiency of this nutrient, and provides a useful alternative (see Chapter 26). Lastly, given the variation in the ability to convert β-carotene to retinol, the use of plasma carotene levels is probably not a useful marker of vitamin A status.36

Vitamin K

In recent years the importance of vitamin K for physiologic functions other than blood coagulation has been recognized, including functions related to both bone and vascular disease. For this reason, the traditional methods of assessment, such as prothrombin and clotting assays, may not be sufficient markers of vitamin K status, at least in regard to these other functions.44 Better functional markers may be serum uncarboxylated osteocalcin (ucOC) levels, or perhaps a serum carboxylated OC to serum total OC ratio. One review suggested that a percent of ucOC greater than 20 is probably optimal, although genetic variations may modify this to some degree.4547

image Conclusion

As described here, many procedures are now available for the assessment of functional vitamin status. Although research continues in this important area, the reader is advised to carefully study the discussion of urinary organic acids profiling (see Chapter 28). Utilizing metabolic products excreted in the urine now allows the clinician far greater specificity in recognizing dysfunctional enzyme systems, whether they are due to genetic deviations or nutritional deficiencies.

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