Vitamin D3

Vitamin D (measured in blood as 25-hydroxyvitamin D) is a fat-soluble vitamin that, in addition to its classic role in bone and dental health, also has immunomodulatory and anti-inflammatory properties[1]. Deficiency (<20 ng/mL) and insufficiency (<30 ng/mL) are common and have been associated with a range of acute and chronic conditions.

However, there is considerable debate about optimal target values[2, 3] . The NICE guideline considers 50–75 nmol/L (approximately 20–30 ng/mL) sufficient, while endocrinologists[4] have recently recommended higher reference values (75–175 nmol/L, or 30–70 ng/mL).

Within Synapse, we align with these higher target values and apply the following cut-offs:

Vitamin B12

There is considerable controversy about optimal cut-off values for vitamin B12. While guidelines generally recommend testing when clear symptoms or anaemia are present, non-specific symptoms such as dizziness, fatigue, and difficulty concentrating are also associated with vitamin B12 deficiency[5]. Since a lack of vitamin B12 can damage the nervous system and increase the risk of conditions such as Alzheimer's disease, vascular dementia, and other neurodegenerative diseases, early detection and correction is of great importance[6].

Vitamin B12 plays a crucial role in various metabolic pathways, which means that functional markers such as methylmalonic acid and homocysteine provide additional insight into effective biological availability. Looking at these functional markers, it has been shown that methylmalonic acid concentrations only reach optimal values[7] at a vitamin B12 level of approximately 250–260 pmol/L (approximately 340–350 ng/L). Synapse therefore applies a higher lower threshold for insufficiency than that advised by the NICE guidelines[8] or the Superior Health Council[9].

Within Synapse, the following reference values apply[10]:

Folate

As with vitamin B12, there is considerable debate[11] about optimal cut-off values for folate. While there is broad consensus on the need for folate supplementation during pregnancy, clear guidelines for the general population outside of pregnancy are harder to find.

To determine the optimal lower threshold for folate, Synapse uses functional markers[12] and applies stricter cut-offs than many existing guidelines. This approach enables early identification of suboptimal folate levels.

The cut-off values[13] we apply are:

Homocysteine

Homocysteine is an intermediate amino acid in the methionine and folate metabolic pathways and serves as a functional integrator of B vitamin status (B2, B6, B9, B12) and choline[22]. In plain terms, it is a natural by-product that builds up when the body lacks certain B vitamins. A deficiency in one or more of these cofactors causes homocysteine levels to rise. In addition to nutritional factors, reduced kidney function (eGFR <60 mL/min) and genetic variants in the MTHFR enzyme (c.677C>T) also contribute to elevated values.

Elevated homocysteine is associated with increased all-cause mortality and with numerous conditions, particularly cardiovascular disease[23]. An independent and partly causal relationship has been described for venous thrombosis, peripheral arterial disease, coronary atherosclerosis, and stroke. The underlying mechanisms include oxidative stress, inflammation, and endothelial dysfunction, with reduced nitric oxide availability. Hyperhomocysteinaemia has also been causally linked to neural tube defects and is associated with cognitive decline and dementia.

Intervention focuses on optimising intake of vitamins B2, B6, B9, B12, and possibly choline or betaine, which can lower homocysteine levels[23, 24] . Homocysteine should be measured in the fasting state, as postprandial methionine intake can cause a transient rise and thereby overestimate the baseline value[22].

At Synapse, we apply the following cut-offs:

Calcium

Calcium (Ca²⁺) is the most abundant mineral in the human body (~1 kg). Approximately 99% is stored in bones and teeth; the remaining 1% circulates freely. This free (ionic) fraction is indispensable for muscle and cardiac contraction, nerve conduction, blood coagulation, enzyme activation, and hormonal signalling. Serum calcium is therefore kept under extremely tight regulation by parathyroid hormone (PTH), active vitamin D, and calcitonin.

Under normal circumstances, calcium levels remain fairly stable. The cut-off values[14] applied by Synapse are:

Magnesium

Magnesium is an essential mineral involved in more than 300 enzymatic reactions in the body. It supports muscle relaxation, nerve impulse transmission, cardiac function (including heart rhythm and blood pressure), bone mineralisation, and energy production. Deficiencies often arise from chronic stress, gastrointestinal disorders, a poor diet, or medication use.

While a low serum magnesium level (hypomagnesaemia) indicates deficiency, a normal level does not rule out depletion; a 24-hour urine measurement can provide additional information. Clinically, muscle cramps, cardiac arrhythmias, fatigue, weakness, difficulty concentrating, and increased stress sensitivity may indicate magnesium deficiency[15].

Under normal circumstances, magnesium levels remain fairly stable. The cut-off values[16, 17, 18] applied by Synapse are:

Zinc

Zinc is an essential trace element found naturally in a variety of foods. It acts as a cofactor for hundreds of enzymes and is indispensable for immune function, protein and DNA synthesis, wound healing, cell division, and signal transduction. It also supports growth and development during pregnancy, childhood, and adolescence, and plays a role in taste perception[19]. Risk factors for deficiency include inadequate dietary intake, chronic alcohol use, and malabsorptive bowel diseases.

In clinical practice, serum or plasma zinc serves as the primary marker, although levels can vary with sex, age, time of sampling, infection, or hormonal changes. The cut-off values applied by Synapse are[20]:

Iron

Iron is a component of haemoglobin and is therefore essential for oxygen transport. It is also found in various enzymes (including cytochromes) and supports energy metabolism, growth, and neurological development. Globally, iron deficiency is the most common nutritional deficiency. It can occur both with anaemia (IDA = Iron Deficiency Anaemia) and without anaemia (IDWA = Iron Deficiency Without Anaemia). Although the latter is often missed, it can have a major impact on quality of life and, in conditions such as chronic heart failure, increases morbidity and mortality[21].

Because a single laboratory parameter can give a misleading picture, Synapse always evaluates an iron profile:

• Transferrin saturation (TSAT): Ratio of bound iron to transferrin.

• Ferritin Reflects iron stores, but rises with (chronic) inflammation.

The table below shows the thresholds we apply:

Ferritin	Male	Female
Iron deficiency	<15µg/L
Possible iron deficiency	<50µg/L Note In the presence of chronic inflammation, ferritin cut-offs are shifted up to a maximum of 100 µg/L to reliably diagnose functional iron deficiency.
Optimal	50-300µg/L	50-200µg/L
Possible iron overload	>300µg/L	>200µg/L

References

1. Walawska-Hrycek, A., Hrycek, E., Galus, W., Jędrzejowska-Szypułka, H. & Krzystanek, E. Does Systematic Use of Small Doses of Vitamin D Have Anti- Inflammatory Effects and Effectively Correct Deficiency Among Healthy Adults? Nutrients 17, 352 (2025).
2. Dietary Reference Intakes for Calcium and Vitamin D. (National Academies Press, Washington, D.C., 2011). doi:10.17226/13050.
3. Holick, M. F. et al. Evaluation, Treatment, and Prevention of Vitamin D Deficiency: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab 96, 1911– 1930 (2011).
4. Amrein, K. et al. Vitamin D deficiency 2.0: an update on the current status worldwide. Eur J Clin Nutr 74, 1498–1513 (2020).5.
5. Aparicio-Ugarriza, R., Palacios, G., Alder, M. & González-Gross, M. A review of the cut-off points for the diagnosis of vitamin B12 deficiency in the general population. Clinical Chemistry and Laboratory Medicine (CCLM) 53, (2015).
6. Lachner, C., Steinle, N. I. & Regenold, W. T. The Neuropsychiatry of Vitamin B 12 Deficiency in Elderly Patients. J Neuropsychiatry Clin Neurosci 24, 5–15 (2012).
7. Muskiet, F. A. J. & Mathus-Vliegen, E. M. H. Commentaar Op Het ‘NHG-Standpunt Diagnostiek van Vitamine-B 12-Deficiëntie’. Ned Tijdschr Klin Chem Labgeneesk vol. 40 (2015).
8. G. Jans & R. Devlieger, C. M. Vitamine B12 - Onmisbaar voor jong en oud. https://www.nice-info.be/nutrinews/vitamine-b12-onmisbaar-voor-jong-en- oud#:~:text=Aanbevolen%20dagelijkse%20hoeveelheid%20vitamine%20B12,4%2C5 %20%C2%B5g%20per%20dag.
9. Hoge Gezondheidsraad (HGR). Voedingsaanbevelingen voor België - 2016. Brussel: HGR; 2016. Advies nr. 9285.
10. Abildgaard, A., Knudsen, C. S., Hoejskov, C. S., Greibe, E. & Parkner, T. Reference intervals for plasma vitamin B12 and plasma/serum methylmalonic acid in Danish children, adults and elderly. Clinica Chimica Acta 525, 62–68 (2022).
11. Pfeiffer, C. M. et al. Applying inappropriate cutoffs leads to misinterpretation of folate status in the US population. Am J Clin Nutr 104, 1607–1615 (2016).
12. Khan KM, J. I. Folic Acid Deficiency. StatPearls [Internet]. Treasure Island (FL): StatPearls (2023).
13. Vos, M. J. et al. Folate reference interval estimation in the Dutch general population. Pract Lab Med 16, e00127 (2019).
14. Office of Dietary Supplements. Calcium Fact Sheet for Health Professionals.
15. National Institutes of Health. National Institutes of Health - Office of Dietary Supplements. Magnesium - Fact Sheet for Health Professionals.
16. Rosanoff, A. et al. Recommendation on an updated standardization of serum magnesium reference ranges. Eur J Nutr 61, 3697–3706 (2022).
17. Yee, J. Magnesium: An Important Orphan. Adv Chronic Kidney Dis 25, 217–221 (2018).
18. Azem, R. et al. Serum magnesium, mortality and disease progression in chronic kidney disease. BMC Nephrol 21, 49 (2020).
19. Qu, X. et al. Serum zinc levels and multiple health outcomes: Implications for zinc- based biomaterials. Bioact Mater 5, 410–422 (2020).
20. National Institutes of Health - Office of Dietary Supplements. Zink - Fact Sheet for Health Professionals.
21. Al-Naseem, A., Sallam, A., Choudhury, S. & Thachil, J. Iron deficiency without anaemia: a diagnosis that matters. Clinical Medicine 21, 107–113 (2021).
22. Refsum, H. et al. Facts and recommendations about total homocysteine determinations: an expert opinion. Clin Chem 50, 3–32 (2004).
23. Smith, A. D. & Refsum, H. Homocysteine – from disease biomarker to disease prevention. J Intern Med 290, 826–854 (2021).
24. Lever, M. & Slow, S. The clinical significance of betaine, an osmolyte with a key role in methyl group metabolism. Clin Biochem 43, 732–744 (2010).