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Archives de pédiatrie
Volume 24, n° 5S2
pages 557-560 (mai 2017)
Doi : 10.1016/S0929-693X(18)30015-0
Laboratory diagnosis of hypophosphatasia
Diagnostic biologique de l’hypophosphatasie

I. Gennero 1, 3, F. Conte-Auriol 3, 4, J.-P. Salles 2, 3,
1 Service de biochimie, institut fédératíf de biologie, hôpítal Purpan, CHU de Toulouse, 31059 Toulouse Cedex 09, France 
2 Unité d’endocrinologie, maladies osseuses, génétique et gynécologie, hôpital des enfants, CHU de Toulouse, TSA 70034, 31059 Toulouse Cedex 09, France 
3 Centre de physiopathologie de Toulouse-Purpan, CPTP, INSERM UMR 1043, université de Toulouse-Paul-Sabatier, 31059 Toulouse, France 
4 Centre d’investigation pédiatrique, INSERM CIC 1436, CHU de Toulouse, 31059 Toulouse Cedex 09, France 

*Corresponding author.

The laboratory diagnosis of hypophosphatasia (HPP) is primarily based on the precise analysis of circulating serum alkaline phosphatase (ALP) activity, determined by biochemical assays. This analysis requires specific conditions of implementation and interpretation and should always be viewed in the light of the clinical and radiological data. Concerns regarding the normal ranges of ALP with respect to age, regarding ALP values that may overlap those of normal subjects in HPP patients, regarding apparently normal ALP values in cases of proven HPP, regarding differential diagnoses that may be responsible for low ALP values outside of HPP will be discussed. High levels of pyridoxal phosphate, a substrate of APL, are of supportive value in the diagnosis of HPP.

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Le diagnostic biologique de l’hypophosphatasie (HPP) repose en premier lieu sur l’analyse précise de l’activité de la phosphatase alcaline (ALP) circulante sérique, mesurée par des méthodes biochimiques. Cette analyse nécessite des conditions précises de réalisation et d’interprétation et doit toujours être confrontée aux données cliniques et radiologiques. Le problème des normes d’ALP en fonction de l’âge, ainsi qu’un recouvrement possible avec les valeurs de sujets normaux en cas d’HPP, de même que les diagnostics différentiels des valeurs basses d’ALP en dehors de l’HPP sont discutés. La question de valeurs apparemment normales d’ALP dans des cas d’HPP prouvée est également discutée. La mesure d’un taux élevé de phosphate de pyridoxal, substrat de l’ALP, est utile pour conforter le diagnostic d’HPP.

The full text of this article is available in PDF format.

ALP refers to a group of glycoprotein enzymes anchored on the surface of the cell membrane that catalyze the hydrolysis of phosphoesters to inorganic phosphate. There are four isoenzymes, three tissue specific at germinal, intestinal and placental level, and a fourth that is nonspecific and ubiquitous (Tissue-Nonspecific Alkaline Phosphatase [TNSALP]). TNSALP is encoded by the Alkaline Phosphatase-Liver (ALPL ) gene located on chromosome 1, and is particularly expressed in bone, the liver and kidneys. HPP (Online Mendelian Inheritance in Man [OMIM] 146 300, 241 500, 241 510) is a rare genetic disease, the severe forms of which are potentially fatal [1, 2]. HPP is caused by mutations in the ALPL gene , which result in weak enzymatic activity and accumulation of non-metabolized substrates. The defective activity of the enzyme results in decreased ALP activity in the plasma. The bone and dental manifestations of the disease are usually in the forefront, contributing to a likely clinical and radiological diagnosis. Genetic confirmation is usually obtained [3]. Laboratory diagnosis, which is based on determining the activity of circulating ALP and certain metabolites or substrates of ALP, is an essential step in confirming the diagnosis. However, the approach is complex and deserves to be known.

Laboratory diagnosis

Diagnosis is primarily based on low serum or plasma ALP activity values. The enzymatic method used to assess the activity consists in the colorimetric method recommended by the International Federation for Clinical Chemistry (IFCC). The method usually measures the rate of hydrolysis of para-nitrophenylphosphate (PNPP) to para-nitrophenol in the presence of Mg2+. The hydrolysis at 37°C, determined by measuring the absorbance of the product at 410/480nm, reflects ALP activity expressed in units per liter (U/L) with a test dependent reference range. It is to be noted that the substrate is not a physiological substrate of the enzyme and that the activity and not the quantity of the enzyme is determined. The sample should be drawn from a tube containing serum or plasma and heparin lithium. Anticoagulants containing ion chelating agents, such as ethylenediaminetetraacetic acid (EDTA) or oxalate are not suitable since the enzyme requires Mg2+as a cofactor. Hemolysis also interferes with the assay.

Even when the sampling and biochemical analysis conditions have been validated, the interpretation of an ALP assay may be complex, particularly with regard to the low values observed in the event of HPP. ALP values depend on age and gender, and are generally between 150 and 300 U/L in neonates. Subsequently, they vary, particularly during puberty. Overall, there is an overlap of the values observed in genetically documented HPP and those of the reference subjects even when age and gender are taken into account. It is therefore important for the laboratory to supply normal ranges for children, by age group, in order to prevent diagnostic errors [4]. It is usually necessary to repeat sampling under standardized clinical conditions. The neonatal period may be particularly problematic [5]. A recent analysis showed that repeatedly low ALP values, in the absence of a patent cause, and even in the absence of clinical symptoms, should give rise to additional clinical, radiological and genetic examinations to investigate for potential HPP [4]. The family survey and samples from relatives, except for the genetic confirmation of an abnormality, are important sources of information. Although a correlation between ALP values and disease severity has been observed, it is difficult to formulate pathogenicity or severity cutoffs [6]. In a given family, low ALP values may be observed in healthy carriers. The genetic abnormality (frequently a mutation with a negative dominant effect) may be present but only clinically relevant in few members of the family. This was the case in a family in which we observed low ALP values in several relatives but only two sisters presented with significant fractures [7]. ALP levels may also be misleading in particular forms of HPP with normal ALP values (pseudo-HPP). Confounding factors may alter the interpretation of the ALP levels such as a fracture consolidation phase or the use of inappropriate normal ranges [1].

There is a bone isoform of TNSALP due to post-translational modifications specific to osteoblasts that may be assayed by an immuno-quantitative method (coupled activity assay), or, simply, an immunospecific method. However, those methods also recognize up to 20% of the hepatic isoenzyme, and are thus of little utility in the diagnosis of HPP.

Pyridoxal phosphate (PLP), the major circulating form of vitamin B6 and the physiological extracellular substrate of ALP, is currently the parameter that contributes most to the diagnosis of HPP, in association with ALP. The dephosphorylation of PLP by ALP appears to be a necessary step in the neuronal uptake of pyridoxal, which is then phosphorylated to PLP in the intracellular medium, where it acts as a cofactor in numerous enzymatic reactions [8]. PLP is involved in various complications of HPP, particularly neonatal pyridoxine-sensitive seizures in severe forms of the disease due to defective neurotransmitter synthesis at neuronal level. The sample for this assay should be drawn from a fasting patient. The plasma or serum should be stored under light protection. Few laboratories conduct the assay by high performance liquid chromatography (HPLC) followed by derivatization and fluorimetric detection. For HPP diagnosis, PLP should be assayed on serum or plasma and not on erythrocytes. The normal ranges are specific to each laboratory and the results may be influenced by an inflammatory state or variation in serum albumin. High PLP values in the absence of vitamin B6 supplementation contribute strongly to HPP diagnosis. Sensitive and specific, ALP levels roughly correlate with disease severity [1]. ALP and PLP values are also usually strongly correlated [9]. Low ALP values and high PLP values are also observed in odonto-hypophosphatasia, a form of HPP whose expression is exclusively odontological [1].

Phosphoethanolamine (PEA) is another potential substrate of ALP. It is not routinely assayed but is mainly used for clinical research. It is usually assayed in the urine by ion exchange chromatography after deproteinization and derivatization with ninhydrin. The issues related to urine collection, the limited availability of the method and the scarcity of published normal ranges are such that the method contributes poorly to HPP diagnosis [10]. The same applies to inorganic pyrophosphate, another physiological substrate of ALP, involved in the pathophysiology of the disease through its inhibitory effect on mineralization. Inorganic pyrophosphate is currently only assayed in research settings.

In children, HPP may be characterized by rickets with high serum calcium and phosphate levels. Calcium and phosphate levels thus provide important complementary diagnostic information especially for infants and in severe neonatal forms. The defective mineralization due to the deficient osteoblastic ALP activity prevents normal calcium and phosphate binding in hydroxyapatite. Thus, in young children, a tendency toward hypercalcemia and hyperphosphatemia is observed, together with hypercalciuria and suppressed parathyroid hormone (PTH) levels. The low PTH values are usually associated with rather high 25-OH-vitamin D due to a defective hydroxylation into 1,25-OH2 -vitamin D. The situation usually normalizes after a few years of life, except if an excessive nutritional calcium/25-OH-vitamin D intake is maintained. Hypercalcemia is rarely observed in older children or adults. Overall, after ruling out the differential diagnoses, repeated low ALP levels, associated with high PLP values, possibly with a suggestive calcium – phosphate profile, should result in genetic testing, and suggest a diagnosis of HPP.

Differential diagnosis of low ALP values

Low ALP values may be observed in a variety of pathological conditions, which are to be identified or ruled out before proceeding to the diagnosis of HPP (Table 1). Investigation for zinc or magnesium deficiency should be conducted if possible. Hypothyroidism and hypoparathyroidism also contribute to lowering ALP. A general evaluation of the metabolic functions, particularly hematological, hepatic and renal functions, is also necessary. Particular conditions such as Wilson’s disease, vitamin D excess, vitamin C deficiency, Cushing’s syndrome and other rare conditions are to be identified. All situations that have marked nutritional impact may lower the ALP, in particular, celiac disease, whose symptoms may be similar to those of HPP [1]. Similarly, prolonged bone resorption treatments (particularly bisphosphonates) diminish ALP levels.

Two situations require a particular attention. First, during the neonatal period, interpretation of ALP values may be difficult, in particular in premature newborns. Second, any factor that alter significantly the infant’s bone mass may be associated to low ALP values such as severe osteogenesis imperfecta (OI).

As obvious and typical radiological criteria for HPP are not always present, PLP assay and precise analysis of the calcium and phosphate profile will then greatly contribute to the diagnosis. It will be critical to identify a tendency toward hypercalcemia, hyperphosphatemia and decreased PTH secretion in this context. Noteworthy, cleidocranial dysplasia, which results in a massive defect of cranial mineralization has a laboratory profile of HPP, including the calcium – phosphate profile; however the bone disorder is usually diagnosed through radiology [11].

Lastly, while erroneous diagnoses of HPP may be made, particularly in adults, by inadequate knowledge of the differential diagnoses, diagnosis may be difficult due to ALP variation throughout life. During puberty, ALP levels rise even in subjects with HPP. Overlap with the values observed in healthy subjects may occur [4, 7]. In addition, the situation may be exacerbated by the elevation of ALP values during fracture consolidation. Moreover, we all know that physicians and laboratory scientists are more prone to reacting to high ALP values than to low values. Normal ranges for ALP assays in pediatric settings have recently been published and may be used to orient standardized or automated assays [12]. In many cases, analysis of the PLP values in the light of clinical and radiological data, with the potential help of the genetic analysis, is essential.


The laboratory evaluation is a key component in the diagnosis of HPP. Diagnosis is essentially based on low ALP levels documented under good conditions by repeated measurements. The essential contributing factors to the diagnosis, in addition to the ALP level, are PLP assay and analysis of the calcium – phosphate profile. The differential diagnostic components for low ALP levels must be considered. The biochemical evaluation constitutes an essential complement to the clinical and radiological data, the family survey and the genetic analysis. It is essential not to delay the diagnosis of severe neonatal and infantile forms whose prognosis depends on rapidly initiated specific management. The biochemistry is also critical to discuss the differential diagnosis and other causes of hypophosphatasemia, which are much more common than HPP.

Statements of interests

I. Gennero and F. Conte-Auriol have no conflicts of interest to report. J.-P. Salles has received fees from Alexion Pharmaceuticals for occasional interventions and expert reviews, and has been or is principal investigator or investigator in clinical trials sponsored by the company.


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