If Hoofbeats are not From Horses, It Could be Zebras!! Isolated Hyper-alkaline Phosphatasemia

Abstract

Alkaline phosphatase (AP) is a membrane bound enzyme and when it is elevated in blood, it is mostly due to either hepatobiliary or bone diseases. We report isolated intestinal hyperphosphatasemia (IAP) in two sisters. Both sisters presented with identical trends of isolated AP elevation. Both underwent extensive workup for liver diseases including cholangiograms, and none was identified. Subsequent isoenzyme electrophoresis showed that 45%–56% of the elevated AP was due to IAP. This elevation of the intestinal AP is consistent with a rare hereditary biochemical abnormality, benign familial intestinal hyperphosphatemia. This condition should be considered in the differential diagnosis of otherwise isolated serum AP levels to avoid unnecessary investigations.

Alkaline phosphatases (APs) are a group of enzymes that include liver, bone and intestinal isoenzymes. When AP is elevated in serum, it is usually due to liver or bone isoenzymes. Benign familial hyperphosphatasemia, also called inherited isolated hyperphosphatasemia, or benign inherited hyperphosphatasemia, is a rare presentation characterized by the presence of elevated AP levels in several members of the same family. This elevation can be due to either intestinal alkaline phosphatase (IAP) alone, or in combination with a lesser elevation of bone or liver AP.^1,2 We describe two sisters who presented with increased levels of serum AP with a preponderance of intestinal isoenzyme in the absence of any known disease. Early recognition of this benign biochemical abnormality, known as benign familial intestinal hyperphosphatasemia (BFIH), will avoid unnecessary investigations and patient's anxiety.

Case 1

A 24-year-old asymptomatic woman was evaluated at our clinic for elevated serum AP found during routine blood tests. She had no risk factors for liver or bone disease and was not on any medications except oral contraceptives. She drank occasionally and never used any recreational drugs. Her younger sister (only sibling) had a similar increase (case 2) in AP. Both parents had consistently normal AP (less than 100 U/L documented between 2015 and 2020), and these were confirmed. Physical examination was normal, liver biochemistry showed normal aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transferase, bilirubin, albumin and total protein. Markers for viral hepatitis, celiac disease and autoimmune liver diseases were absent (Table 1). Ultrasound of the liver was normal, but a magnetic resonance cholangiopancreatography (MRCP) was reported as showing evidence for primary sclerosing cholangitis (PSC) with a possible stricture in the common hepatic duct and intrahepatic ductal beading. However, endoscopic retrograde cholangiopancreatography (ERCP) was normal, and duodenal biopsies did not show any evidence of celiac disease. When the initial MRCP was reviewed by a radiologist with expertise in MRCP, that test was interpreted as normal. The patient also underwent a colonoscopy to assess for possible inflammatory bowel disease; mucosal appearances were normal and multiple random biopsies from the entire colon showed normal histology. The fractionation of AP indicated that 56% (ranged from 56 to 75% on three separate occasions) came from the intestine. The elevation of isolated AP persisted (∼11 years, Figure 1) until the time of reporting, but the patient remains asymptomatic and with excellent health.

Table 1.

Liver Biochemical Characteristics and Diagnostic Tests.

Test		Case 1	Case 2
Alkaline phosphatasea	Total (U/L, NR 33–115)	323	302
	Intestinal % (NR 1–24%)	56	45
	Bone % (NR 28–66%)	32	37
	Liver % (NR 25–69%)	13	18
Aspartate aminotransferase (IU/L)		17	15
Alanine aminotransferase (IU/L)		16	10
Gamma GT (IU/L)		19	22
Anti-mitochondrial antibody		Negative	Negative
Anti-smooth muscle antibody		Negative	Negative
Anti-LKM		Negative	Negative
Alpha-1-antitrypsin phenotype		MM	MM
TSH (mIU/L)		2.86	3.60
Blood type		A+	A+
Celiac serology		Negative	Negative
Duodenal biopsy		Normal	Normal
Cholangiogram (ERC)		Normal	Normal

Gamma GT, gamma-glutamyl transferase; anti-LKM, anti-liver-kidney microsome antibody; TSH, thyroid-stimulating hormone; ERC, endoscopic retrograde cholangiography.

^aQuest Diagnostics/Nichols Chantilly–Chantilly PA.

Figure 1.

The graph shows alkaline phosphatase levels over the period of 10 years in case 1 (age 24–34 years) and sister case 2 (age 22–31years).

Case 2

A 22-year-old healthy woman (younger sister of case 1) also was seen for elevated serum AP. She had similar history as her sister and drank only occasionally and never used any recreational drugs. Except for the AP (three times of normal limit), her liver biochemistry was normal. Other tests including markers for viral hepatitis, celiac disease and autoimmune liver disease were negative (Table 1). MRCP was reported as showing beading of intrahepatic ducts suspicious for PSC, but ERCP was essentially normal as was a duodenal biopsy for celiac disease. As in case 1, a subsequent normal interpretation of the MRCP was provided by an experienced radiologist. Colonoscopy was also normal with normal mucosal histology. AP isoenzymes showed that 53% was from the intestine. Just like her sister, she maintained excellent health although elevated AP persisted with an upward trend with time as shown in Figure 1.

Discussion

Usually, an elevated serum AP level in clinical practice is due to hepatobiliary disorders, drug-induced liver injury or abnormal bone metabolism. However, AP can also be elevated in physiological conditions such as in pregnancy, transient hyperphosphatasemia of infancy, and during normal growth spurts. It can also be elevated in diabetes mellitus and more commonly in cirrhosis or chronic kidney diseases (CKDs).^{3, 4, 5} The two sisters we presented here had no evidence of any such conditions, and represent a relatively rare condition called BFIH, due to their benign course, predominance of the intestinal AP isoenzyme and the occurrence of an identical presentation in two siblings. Based on consistently normal ALP levels in both parents, we suspect that the inheritance pattern of this disorder in these two sisters is autosomal recessive.

Few case reports have appeared in the literature with familial clustering of subjects with benign elevation of AP, but in many of them the increased AP represented mostly the intestinal fraction.^2,6,7 AP is a ubiquitous membrane-bound enzyme. It is encoded by four genes—tissue non-specific AP (TNAP), intestinal AP (IAP), placental AP and germ cell AP. While TNAP is expressed in multiple tissues, including the liver, bone, kidney, colon and leukocytes, IAP is mostly expressed and secreted by the small intestinal epithelial cells.^8,9 A detailed discussion of the role of IAP is beyond the scope of this brief article, but it has been thoroughly reviewed elsewhere.⁸ IAP plays an important role in gut homeostasis including mucosal defense against inflammation and maintenance of the gut microbiome.^{9, 10, 11, 12, 13, 14, 15}

There are two forms of IAP, membranous and soluble/secreted forms.^16,17 Studies in the rat have identified two distinct mRNAs encoding nucleotide sequences for these respective IAP forms.¹⁸ Additionally, the rat membranous and soluble/secreted IAP forms are distinct based on differences in their fucose and N acetyl galactosamine content, and mobility on electrophoresis, isoelectric point and the observation that there are no changes in the percentage of soluble isoenzyme on addition of proteases.¹⁹ The membranous type is located in the brush border of the duodenum, and it has a slow turnover and a larger size compared with the secreted type, also called soluble AP, which has a shorter half-life (25 min).¹⁷ IAP is released from the brush border membrane into the lumen. After high-fat diet feeding in rat, it has been shown that there is > 10-fold release of IAP into the intestinal lumen resulting in depletion of IAP in the brush border membrane. This is followed by progressive mRNA increase and ultimate restoration of IAP by 6-h in the brush border membrane.²⁰ Alternatively, other studies have demonstrated the release of IAP by the action of phospholipase C or D, cleaving the attached glycosyl—phosphatidylinositol (GPI) anchor, allowing IAP to enter into the circulation in a soluble form.^21,22

Approximately 0%–24% of serum total AP is represented by the intestinal isoenzyme.^23,24 A modest increase in the IAP isoenzyme has been reported in seemingly healthy fasting individuals with blood groups O or B.²⁵ It has also been noted that a high fat diet can cause notable significant elevation in serum IAP levels for up to 15 h, with maximal activity at 7 h. In this study in rats, the levels of soluble intracellular IAP in the intestinal mucosa remained unchanged, despite a 3.5-fold rise in serum IAP.²⁶ Similarly in humans, increased secreted ALP activity occurs more often in blood group B or O than in most other blood types, and this increase usually peaks within 3–6 h after a high fat meal.²⁷ If AP is marginally increased in any given patient with no obvious cause for such elevation, it may be prudent to retest serum AP in the fasting state. After a high fat diet, the intestinal lymphatics deliver IAP into the circulation, resulting in the elevation in serum IAP levels. Additionally, it was observed that serum IAP most likely represents soluble IAP due to its structural similarities on isoelectric focusing. Hence, it has been suggested that the soluble IAP is independently regulated, and the elevation in serum IAP originates from the pool of soluble enzyme in the intestinal mucosa.²⁶ Stimuli other than a fat-rich diet (including cholecystokinin and bethanechol) have also been shown to increase both intestinal luminal IAP and serum IAP levels simultaneously in the rat, suggesting a complex regulation for secretion into the serum.²⁸

Serum IAP can be elevated in patients with diabetes mellitus, cirrhosis or chronic renal failure.^{3, 4, 5,9} In a study of 189 patients with diabetes or cirrhosis, an ‘intestinal variant AP’ was found on electrophoresis in about one in four patients with elevated serum IAP.⁴ Like IAP, this variant band was seen more frequently in blood groups O and B. However, it was noted that the variant AP was seen only in the presence of IAP, and in contrast, IAP could be seen without the variant AP. Based on this, it was concluded that this variant AP likely represents IAP and is regulated by similar physiological mechanisms.⁴ In CKDs, non-specific elevation of IAP may be rarely seen. Here, IAP has been shown to be present in kidney brush border membranes in up to 8% to 21% of examined specimens.⁵

In contrast with these mechanisms for increased secretion or production of IAP in the presence of chronic disease, hyperphosphatasemia is a group of disorders characterized by a series of mutations in different parts of the ALP gene. One of these disorders is BFIH. Various mechanisms have been proposed for the pathogenesis of BFIH including a mutation affecting the gene that encodes for the intestinal isoenzyme, ALPI. A mutation could occur in the extramembranous part of the protein, or in the portion of the IAP where the GPI C-terminal addition is inserted, thus creating the lipid extension to bind ALP to the membrane.²⁹ Ishige et al, sequenced the ALPI gene from one patient with BFIH and examined the differential expression of soluble and membrane-bound fractions of IAP after transfection into NIH3T3 cells. They reported a deletion–insertion variant in the C-terminal end of the IAP coding region, preventing addition of the GPI linkage.³⁰ In the cultured cells, there was a relative increase in the fraction of soluble IAP expression compared with membrane-bound IAP. Based on these observations, the authors postulated that elevation of soluble IAP in this case of BFIH was perhaps due to attenuation of the membrane binding capability. Unlike mutations in the catalytic region of ALPI when IAP activity is lost, resulting in severe IBD,³¹ the mutation seen in BFIH preserves activity, affects only the intestinal isoenzyme, and leads to increased serum activity without any clinical consequences.³¹ Even though the case reported by Ishige et al. provides a novel underlying mechanism for BFIH, it will be necessary to investigate the genomic and molecular mechanism in other affected patients to determine whether this proposed mechanism is present in all or most cases of BFIH.

The two cases presented here illustrate the importance of considering BFIH in the differential diagnosis when asymptomatic patients without suspected or known liver or bone disease present with an isolated elevation of serum AP. Before performing invasive tests, it is prudent to check ALP isoenzymes in such subjects with isolated elevation in ALP, especially when multiple family members are affected. Early recognition may avoid invasive tests and delay in making a firm diagnosis. It is also important to collect serum from as many members of the family as possible, to obtain DNA for sequencing and to learn more about the inheritance pattern in BFIH. In these two siblings, it is more than likely that the inheritance is autosomal recessive in nature.

CRediT authorship contribution statement

Mahak Chauhan: Writing - original draft, Final approval of the version to be submitted. David H. Alpers: Writing - review & editing, Final approval of the version to be submitted. James P. Hamilton: Writing - review & editing, Final approval of the version to be submitted. Paul J. Thuluvath: Conceptualization, Methodology, acquisition of data, Formal analysis, interpretation of data, Final approval of the version to be submitted.

Conflicts of interest

The authors have none to declare.

Funding

None.