Case series demonstrating the effectiveness of Zn supplements in adults with hypophosphatasia who were overlooked for a long time

Abstract

During routine medical examinations, we incidentally diagnosed 11 individuals with hypophosphatasia, which is a condition that has previously gone unrecognized. Six patients also had low levels of serum Zn. Supplemental Zn treatment was effective in controlling the symptoms of hypophosphatasia in all patients. One patient complained of muscle pain and twitching after starting Zn supplement treatment temporarily. The subjected three patients in whom Zn supplements were effective showed improvements in bone mineral density and general malaise. Zn supplements might be useful for controlling symptoms in patients with hypophosphatasia who have mild symptoms.

Introduction

Hypophosphatasia (HPP) is a rare metabolic disorder characterized by a deficiency in the activity of alkaline phosphatase (ALP), which is an enzyme essential for the proper formation of bones and teeth. Dysfunction of ALP, which is caused by genetic mutations, leads to impaired mineralization, and subsequent weakening of skeletal structures and dentition.^1,2 ALP is broadly expressed in various organs, including the liver, kidneys, and small intestine, as well as in osteoblasts. It is predominantly localized to the cell membrane and is then subsequently released into the circulation. As ALP is expressed mainly in the liver and bones, increased serum ALP levels typically reflect pathological processes, such as bone fractures or hepatic injury. In patients with HPP, serum ALP levels are characteristically low. HPP is classified into six subtypes, with the most severe form, namely, the perinatal type, occurring in ~1 in 150,000 live births. The carrier frequency of pathogenic mutations in ALP is estimated to be ~1 in 600 in Japan, ³ and may reach as high as 1 in 100 in certain other country.^4,5 In patients with milder forms of HPP, clinical manifestations are often minimal. Moreover, patients with HPP may present with normal ALP levels following bone fractures, and children typically have higher baseline ALP levels than adults owing to ongoing skeletal growth. Consequently, mild cases of HPP are frequently overlooked until adulthood, whereas awareness of HPP has increased among osteoporosis specialists, recognition of the condition remains limited among other specialists and primary care physicians. In a previous study, ~50% of individuals with HPP had previously experienced at least one fracture. ⁶ In our cohort, we identified 11 patients who were incidentally diagnosed with HPP during routine medical health checkups, and the majority of them had a history of recurrent fractures. Some also had low levels of serum Zn. In this study, we analyzed the disease course after Zn treatment of four patients with HPP who showed low levels of serum Zn, to clarify the efficacy of Zn treatment for HPP.

Levels of serum Zn in patients with HPP

In clinical practice, adult HPP is screened based on the following criteria: (1) persistently low serum ALP levels; (2) a history of fractures, chronic fatigue, or unexplained joint or muscle pain; and (3) a family history suggestive of hereditary involvement, even if the individual is asymptomatic. Relevant family history findings include the following: (a) premature loss of deciduous teeth (typically between 1 and 4 years of age); (b) short stature; (c) gait abnormalities; and (d) a tendency of frequent fractures.

Between September 2022 and August 2023, 13,550 people (8607 men and 4943 women) in our health checkup center were screened for HPP. A total of 79 people (49 men and 30 women) met the ALP criteria, which is ALP values below 33 U/L for men and below 29 U/L for women (ALP general standard: 38–113 U/L for both men and women). A written recommendation to undergo further detailed medical evaluation was provided to these individuals. Subsequently, 13 individuals (six men and seven women) presented to the genetic outpatient clinic. Among them, 11 were found to have genetic mutations in ALPL, and clinical features consistent with HPP,^7,8 including a family history of short stature. Serum Zn and 25-hydroxyvitamin D levels were measured in the patients diagnosed as having HPP, as previously reported by our group. ⁹ Clinical manifestations associated with HPP were mild or absent in these patients. The number of fractures experienced since childhood ranged from none to 10, with no notable issues in fracture healing. Of the 11 patients, six had low serum Zn levels, and five out of the seven assessed patients showed decreased serum 25-hydroxyvitamin D concentrations.

Disease courses of the patients after treatment with Zn supplements

Case 1

The patient was a 29-year-old woman who was found to have abnormally low levels of ALP at a health checkup (Figure 1). She complained of general fatigue without arthralgia or muscle pain. Her teeth were partially broken, and she also had dysmenorrhea. Regarding her past medical history, she had experienced two bone fractures at a young age. Regarding her family history, her father, elder sister, grandfather, and grandmother had all experienced bone fractures. Her ALP and Zn levels were low, at 25 IU/L and 55 μg/dL, respectively. Genetic analysis of ALPL showed that she had the 529G>A mutation, which is considered to be pathogenic. Her bone mineral density (BMD) Z-scores were calculated for the lumbar spine, femoral neck, and whole body. Her BMD of the lumbar spine and left femoral neck were 1.012 and 0.861 g/cm², respectively. T-scores of the lumbar spine and left femoral neck were −1.3 (85%) and −0.8 (90%), respectively. After starting Zn medication (NOBELZIN^®; 50 mg/day: Nobelpharma Co., Ltd.), her serum Zn and ALP increased to 76 μg/dL and 35 U/L, respectively (Figure 1). The patient experienced no fractures while taking Zn supplements. However, her general fatigue without muscle pain still continued. Therefore, the dosage of the Zn medication was increased to 75 mg. Subsequently, her symptoms improved temporarily. The T-scores of the lumbar spine and left femoral neck became −1.3 (84%) and −0.8 (90%), respectively. BMDs of the lumbar spine and left femoral neck were 0.999 and 0.865 g/cm², respectively. Her trabecular bone score (TBS) of the lumbar spine improved from −1.5 before treatment to −1.2 at 10 months after the initiation of treatment. There were no side effects, including decrease in Cu and Ca levels during the 10 months (Table 1).

Figure 1.

Serum Zn and ALP levels both before and after treatment with Zn. In all cases, both serum and ALP increased after Zn treatment after 3 months.

ALP: alkaline phosphatase.

Table 1.

Characteristics of the patients with HPP.

Case	Age (years)	Sex	Complaint	Number of fractures	ALP (U/L)	Serum Zn (µg/dL)	BMI lumbar spine/femur (%)	Genomic study of ALPL mutation (all heterozygous)	Diagnosis	Date of start of Zn supplementation	Duration of follow-up (months)
1	29	F	Muscle pain Migraine	1	25	55	85/90	c.529G>A	Adult-type HPP	July, 2024	10
2	49	F	Muscle pain Migraine Arthralgia (knee joints)	0	19	56	110/87	c.529G>A c.1559del	Adult-type HPP	June, 2023	22
3	49	M	None	10	28	59	87/73	c.1171dup	Pediatric-type HPP	April, 2023	24
4	37	F	Migraine Arthralgia (hip joint)	1	29	51	118/108	c.529G>A	Adult-type HPP	May, 2023	3

HPP: hypophosphatasia.

Case 2

The patient was a 49-year-old woman who was found to have abnormally low levels of ALP at a health checkup. She complained of general fatigue, muscle pain and arthralgia of the fingers, hip, knee joints, and lumbar. Her teeth were normal. Her past medical history included two bone fractures at a young age. Her family history included bone fractures of the father, elder sister, grandfather, and grandmother. Her T-scores were equivalent to people of 50 years of age. BMDs of the lumbar spine and left femoral neck were 1.311 and 0.857 g/cm², respectively. T-scores of the lumbar spine and left femoral neck were +0.8 (110%) and −1.0 (87%), respectively. Her serum Zn level was low, at 56 μg/dL and hence, Zn treatment was started. Analysis of the ALPL gene showed heterozygous 529G>A and 1599del mutations, which are both pathogenic. The absence of bone calcification was confirmed using an X-ray. After starting Zn supplements (50 mg/day), her serum Zn level increased to more than 250 μg/dL. However, twitching of her upper and lower extremities appeared, without any changes in the levels of iCa and Ca. She hence continued taking the medication in minimum dose (25 mg/day). Her BMDs of the lumbar spine and left femoral neck were improved as 1.350 and 0.867 g/cm², respectively. T-scores of the lumbar spine and left femoral neck were + 1.1 (113%) and −0.7 (90%), respectively, at 9 months after the initiation of treatment. There were no side effects during the 22 months of follow-up (Table 1).

Case 3

The patient was a 49-year-old man who was found to have an abnormally low level of ALP of 37 IU/L at a health checkup. His past medical history included 10 bone fractures since a young age, without any particular diagnosis. His teeth were normal. His family history showed no fractures, arthralgia, or kidney disease. His T-scores were equivalent to those of people of 50 years of age. BMD of his lumbar spine and left femoral neck were 0.995 and 0.759 g/cm², respectively. T-scores of the lumbar spines and left femoral neck were −0.9 (87%) and −2.1 (73%), respectively. His serum Zn level was low at 59 μg/dL, and Zn treatment was started.

ALPL gene analysis demonstrated a heterozygous mutation of c.1171dup (p.Arg391ProfsTer14), which is pathogenic. No bone calcification was observed on X-ray analysis. After starting Zn supplements (50 mg/day), he had no fractures, and there were no side effects for 24 months. After starting treatment, his Zn and ALP levels increased to 158 μg/dL and 37 IU/L, respectively. His TBS also increased slightly. BMD of the lumbar spine and left femoral neck were 1.012 and 0.764 g/cm², respectively. T-scores of the lumbar spine and left femoral neck were −0.8 (89%) and −2.1 (73%), respectively. His TBS also increased from 1.338 to 1.373 at 6 months after the initiation of treatment (Table 1).

Case 4

The patient was a 37-year-old woman who was found to have an abnormally low level of ALP of 29 IU/L at a health checkup. Her past medical history included bone fracture once and fatigability, without any particular diagnosis. T-scores of the lumbar spines and left femoral neck were −0.9 (109%) and −2.1 (96%), respectively. Her serum Zn level was low at 51 μg/dL, and Zn treatment was started. ALP and Zn levels increased promptly at 3 months after the initiation of treatment, without any symptoms (Figure 1). Her symptoms did not change during the treatment, a second BMD check was not done.

We reviewed each patient’s history and laboratory data for possible secondary causes, such as malnutrition, malabsorption, and medication use (e.g. proton pump inhibitors), and found no lines of evidence suggesting that these factors were involved.

Discussion

Adult HPP often manifests with nonspecific clinical features, such as osteopenia, osteomalacia, and muscle involvement, leading to frequent misdiagnosis as osteoporosis during routine evaluations. ¹ The predominant symptoms observed in adult HPP patients include musculoskeletal pain, reported in 100% of patients, and muscular weakness, present in ~83.3% of patients. ¹⁰ In this study, we identified patients with adult HPP who were overlooked in regular checkups because of their mild symptoms.

Enzyme replacement therapy with asfotase alfa has demonstrated substantial efficacy in the treatment of HPP, leading to improvements in skeletal mineralization, mobility, and overall quality of life in patients across various age groups.^11–13 Although HPP treatment is covered by insurance in Japan, the cost is high and its safety has only been confirmed for 5 years. ¹⁴ Moreover, the efficacy of asfotase alfa for kidney stones and renal failure have not been not confirmed. Therefore, new treatments other than enzyme treatments are needed to control HPP. In this study, all patients showed low levels of vitamin D. It has been reported that oral vitamin D supplementation is not associated with reduced fracture risk or increased bone mineral radial density in a multicenter double-blind randomized trial. ¹⁵ Half of the patients showed low levels of serum Zn, but there were no other notable laboratory findings, including abnormalities in Hgb (Hemoglobin), MCV (Mean Corpuscular Volume), and MCHC (Mean Corpuscular Hemoglobin Concentration). Zn is a cofactor of tissue-nonspecific ALP, and vitamin D plays a pivotal role in regulating bone matrix mineralization. Previous studies have indicated that the prevalence of Zn and vitamin D deficiencies in patients with HPP is comparable to that observed in the general population. ¹⁶ Zn supplement administration did not cause any side effects in this study.

Zn deficiency is thought to predispose patients to osteoporosis. It has also been shown that taking Zn can increase bone mass by 0.5% in 6 months, and by as much as 2.5% if continued for a year. ¹⁷ Bisphosphonates are used as first-line drugs for the treatment of osteoporosis. For patients with HPP, bisphosphonates have a negative effect of bone formation. ¹⁸ According to Fung et al., patients with thalassemia frequently have a low plasma Zn level, which has been associated with low bone mass. They showed that in 42 young patients (aged 10–30 years) with thalassemia, Zn supplementation resulted in a greater gain in total body bone mass than did placebo using bone mineral content and areal BMD. ¹⁹ Sadighi et al. reported that the administration of Zn causes a significant increase in serum Zn and ALP activity, and substantial progression in callus formation on bone X-ray in 60 patients with traumatic bone fracture compared with the controls. ²⁰ Zn has been shown to stimulate gene expression of the transcription factor Runx2, which is associated with osteoblastic cell differentiation. In addition, the oral administration of Zn acexamate has been shown to have a substantial restorative effect on bone loss in various pathophysiologic conditions, including aging, diabetes, and fracture healing. ²¹

All four patients in this study showed increased serum ALP and Zn levels, without a decrease in Cu and Fe levels. The clinical efficacy of Zn supplements was obscure because of the lack of a standard protocol for Zn treatment, and variations in the durations of follow-up. In the meantime, Zn supplement treatment did not cause arthralgia, bone fracture, or fatigue in any of the patients. In this study, Zn levels decreased after treatment, without a normalization of ALP level. Research on Zn levels in osteoporotic women is scarce. Mahdaviroshan et al. performed a randomized, double-blind, placebo-controlled clinical trial to assess the effect of Zn supplementation on serum Zn and calcium levels in postmenopausal osteoporotic women. A total of 60 women who were referred to a rheumatology clinic were randomly divided into the intervention (220 mg Zn sulfate daily) and placebo groups. In the intervention group, serum Zn levels were significantly higher after 60 days of treatment, whereas serum calcium levels remained unchanged. They concluded that postmenopausal osteoporotic women may benefit from Zn supplementation. ²² The review included 16 studies and concluded that Zn levels appear to be lower in subjects with bone metabolism-associated pathology. Regarding daily Zn intake, a high proportion of the population (more than 20%), appears to be at risk of having inadequate Zn intake. ¹⁷ The literature suggests that insufficient Zn intake (<3 mg/day) could be a risk factor for bone fractures and for the development of osteopenia and osteoporosis. Zn supplementation (40–50 mg/day) could have beneficial effects on bone health in terms of maintaining BMD and faster healing in the event of fractures, with even more favorable results in situations of reduced Zn intake from food.

Wiedemann et al. analyzed variant type and data in a cohort of 263 HPP patients. ¹⁶ Of this cohort, 73.5% had vitamin D deficiency and 27.2% had Zn deficiency similar as were previously described for the general population. ²³ The elevated requirement for Zn in patients with HPP may also contribute to altered serum levels. The effects of Zn and vitamin D supplementation in patients with HPP who were deficient in these nutrients were evaluated retrospectively. Specifically, 10 patients with Zn deficiency and 38 patients with vitamin D deficiency received supplementation following general clinical guidelines. The interventions led to significant increases in serum Zn and 25-hydroxyvitamin D levels. However, the levels of other disease-associated biomarkers, including ALP and pyridoxal-5′-phosphate, as well as bone turnover markers, such as phosphate, calcium, parathyroid hormone, bone-specific ALP, creatinine, and deoxypyridinoline, remained unchanged. These findings suggest that standard supplementation protocols for Zn and vitamin D can be safely applied to HPP patients to correct their deficiencies without adversely affecting bone metabolism or calcium homeostasis. ¹⁶ Zn promotes osteoblast differentiation by stimulating Runx2 expression, and increases BMD. As standard osteoporosis treatments, such as bisphosphonates, are contraindicated in HPP, and enzyme replacement therapy is costly with limited long-term safety data, Zn provides a safe, affordable alternative. Additionally, Zn deficiency is common in the general population and is associated with increased fracture risk, which also supports its supplementation in HPP patients.

In this study, the responses against Zn supplementation were different among the patients. Symptoms and BMD or TBS improved in three out of the three patients (one did not check secondary BMD). Each patient had a different genotype. As the high genetic variability of ALPL results in high clinical heterogeneity,^24,25 their different responses to Zn treatment might be owing to their different ALPL gene variations. To our knowledge, there have been few trials to date of Zn supplementation in HPP patients. HPP is a rare genetic metabolic bone disease that can cause chronic pain and fractures. However, recent genomic analyses have demonstrated that there are more cases of mild forms of HPP than were previously recognized. The hallmark of this mild form is a persistently low serum ALP level. To not overlook such cases of mild HPP, it is important for physicians to recheck ALP levels when they encounter patients with fractures from minor traumas or patients with frequent fractures. Further clarification of the details of this disorder is needed. Other limitations of this study include the lack of a clearly defined duration of Zn supplementation or follow-up period. This may have affected the ability to accurately evaluate the long-term efficacy and safety of Zn treatment. In addition, there is the possibility that the changes in TBS and BMD may be within the range of measurement variability. Future statistical studies with standardized treatment durations and systematic follow-up schedules are needed to more accurately assess the clinical response of Zn supplementation in HPP patients.

Conclusion

Zn supplements might be a promising treatment for controlling symptoms in patients with HPP who have mild symptoms. Future statistical studies with standardized treatment durations and follow-up schedules are required to reach a clear conclusion.