Plasma phylloquinone concentrations increase following acupoint injection for primary dysmenorrhea

Abstract

Therapeutic benefits of acupoint injection of vitamin K in Spleen-6 for the treatment of primary dysmenorrhea have been observed in limited clinical settings. However, menadione, the form of vitamin K most studied for treating dysmenorrhea, is not routinely used in clinical practice in North America. As part of a larger clinical trial among women with primary dysmenorrhea aged 18 to 25 years old, we conducted a sub-study to test the plasma concentration of phylloquinone (vitamin K₁). We collected blood samples of four women prior to and 24-48 hours following acupoint injection of phylloquinone in Spleen-6. Despite rapid turnover of phylloquinone observed in prior studies, we found that plasma phylloquinone concentrations significantly increased from pre-injection to one to two days after injection. Interestingly, higher phylloquinone was correlated with less pain intensity among women with dysmenorrhea. Additional research is needed on the association between vitamin K and menstrual pain, including the role of vitamin K deficiency in inflammation and pain, and the possible mechanisms of acupoint injection of vitamin K for the treatment of primary dysmenorrhea.

Introduction

Acupoint injection of vitamin K in Spleen-6 (SP6) has been used as an effective treatment for primary and secondary dysmenorrhea, and pelvic pain for the past three decades in local clinical settings in China and Italy [1-4]. Therapeutic benefits of this treatment include rapid pain relief within 30 minutes, less restriction in daily activities, reduced number of hours in bed, and a decrease in pain medications used. SP6 is a point frequently used for regulating the menstrual cycle [5], but the use of vitamin K for menstrual-related conditions is relatively uncommon. The vitamin K form studied to date in terms of acupoint injection is menadione (vitamin K₃), also referred to as menadiol (vitamin K₄) when injected in a water soluble form. Menadione is no longer used therapeutically in the United States due to concerns of liver damage [6]. However, recent data suggest that menadione is an intermediate in the conversion of the primary dietary form, phylloquinone (vitamin K₁) to another vitamin K form, menaquinone-4 [7]. In the United States, vitamin K is administered in the form of phylloquinone to infants to prevent hemorrhage and to adults on anticoagulant therapy to reverse the effects of under-coagulation. The functions of vitamin K beyond its role in coagulation are increasingly of interest [8, 9], but little is known about the potental mechanism of vitamin K on menstrual pain. Prior research suggests that vitamin K deficiency is associated with menstrual disorders [10]. In addition, studies using animal models indicate relaxation of the uterine muscle spasm of rabbits and rats within three to five minutes of menadione administration [11].

Plasma phylloquinone concentrations administered orally or intravenously peak and return to close to baseline levels, usually within 24 hours [12]. Intramuscular injection of vitamin K may have a depot effect producing slow absorption from the site of injection [13]. This depot effect may explain the prolonged benefits of acupoint injection suggested by prior research [1], although menadione would have a different pharmacokinetics compared to phylloquinone based on differences in biochemical structure and depending on the solubility of the form injected. To our knowledge, empirical evidence of plasma concentration following acupoint injection of phylloquinone has not been collected. To address this gap, we analyzed plasma phylloquinone concentration following acupoint injection treatment among women with dysmenorrhea.

Materials and Methods

All study procedures were approved by the University of California, San Francisco (UCSF) Committee on Human Research and the UCSF Clinical Research Center and registered in ClincalTrials.gov (NCT00995917). We conducted a pilot clinical trial in the U.S. to assess the acceptability, feasibility and efficacy of acupoint injection of phylloquinone in SP6 among young women with primary dysmenorrhea. The study consisted of a randomized cross-over pilot comparing phylloquinone in SP6 with saline in a non-acupuncture point [14]. Here we describe a sub-study conducted to determine plasma concentration of phylloquinone before and after acupoint injection.

Detailed methodology is previously described [14]. Recruitment primarily occurred through outpatient clinics at local community health centers in San Francisco. Study staff pre-screened prospective participants by telephone based on inclusion and exclusion criteria. We consented and enrolled study participants who were English-speaking young women, 18 to 25 years of age, with primary dysmenorrhea defined as recurrent painful periods not fully relieved by other treatment for at least six months. We excluded women who had a history of term pregnancy, abdominal surgery, pelvic inflammatory disease or chlamydia; or current pregnancy, diagnosed or suspected acute, chronic conditions, concomitant therapy for acute or chronic pain, use of hormonal contraception, treatment with anti-coagulant drugs for any reason, intolerance to nonsteroidal anti-inflammatory drugs or aspirin, bleeding or non-cyclic pelvic pain, or dysmenorrhea due to any other suspected or recognized causes. Diagnosis of primary dysmenorrhea was confirmed by the nurse practitioner based on history and physical examination. All consent, screening and study procedures were conducted at the UCSF Clinical Research Center (CRC). Participants were offered an incentive of $40 for the injection visit, $30 for their first blood draw and $50 for their second blood draw.

Two nurse practitioners trained in acupoint injection performed study procedures. Injection treatment was administered to study participants presenting in pain, during the first 2 days of their menstrual cycle. Acupoint injections were administered bilaterally in both legs in SP6, located on the lower leg, three cun (the width of a person’s thumb at the knuckle) proximal to the peak of the medial malleolus. This area is just along the posterior aspect of the tibial bone. The injected muscle was the soleus. The nurse pracitioner inserted a No. 23 gauge 2.5 cm long needle into the point and 5 mg of phylloquinone per 0.5 ml volume was injected intra-muscularly. The needle was removed, and the point patted with gauze pads if bleeding was present. Consistent sourcing, quality, and storage of phylloquinone were ensured by UCSF Pharmacy Services.

Blood samples

To assess plasma phylloquinone concentration, each participant had two blood samples drawn. The first blood draw took place either immediately after the participant was consented or at another scheduled time prior to acupoint injection of phylloquinone in SP6. The second blood draw took place 20-48 hours after the acupoint injection treatment. Participants were instructed to fast 12 hours before each blood draw. Nurses at the CRC confirmed whether participants had fasted based on verbal self-report and then drew ten cc’s of blood for each blood sample. The blood was spun and stored in a freezer at -70 C at the CRC until shipped to the Vitamin K Laboratory at the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University for analysis of phylloquinone content. Plasma concentrations of phylloquinone were measured by reverse-phase high-performance liquid chromatography, which has a lower limit of detection of 0.02 nmol/L for phylloquinone [15].

Pain intensity

The study participants’ level of menstrual pain was measured using an 11-point (0-10) pain intensity numerical rating scale (PI-NRS) [16]. At the injection visit, PI-NRS was obtained immediately before injection of vitamin K1 (defined as baseline), and then 5,15, 30 and 60 minutes after the injection treatment. The difference in PI-NRS from baseline to 60 minutes was used as the primary clinical outcome in the study. At the consent visit and at monthly follow-ups, we obtained participants’ PI-NRS of their worst pain from cramps during their last menstrual period. We used paired Student’s t-tests to assess the statistical difference of phylloquinone concentrations and pain intensity before and after acupoint injection. We also assessed the correlation between worst pain intensity from cramps during the most recent menstrual cycle and phylloquinone using Pearson’s correlation.

Results

We recruited four women to participate in the substudy of plasma phylloquinone concentration. All four participants were non-Hispanic white and had obtained a college level of education. Participants were on average 22 years of age and had a current cycle length of 29 days. Average age of menarche was 13 years.

All four participants had notable changes in plasma phylloquinone concentrations from pre- to post-injection of phylloquinone. Analysis of plasma phylloquinone indicated non-detectable concentrations before acupoint injection. In contrast, phylloquinone concentrations ranged from 40 to 425 nmol/L, 20-48 hours following injection (see Table 1).

Table 1.

Plasma phylloquinone concentrations before and after acupoint injection of phylloquinone in Spleen-6

Participant	Phylloquinone at baseline (nmol/L)	Phylloquinone after injection (nmol/L)	Number of hours after injection blood sample was drawn
A	ND	130	46
B	ND	40	48
C	ND	425	22
D	ND	190	21

Notes. ND = Not Detectable (<0.02 nmol/L)

On average, participants had a baseline pain intensity of 4.63 immediately before acupoint injection of phylloquinone in SP6. Sixty minutes following injection, average pain intensity decreased to 1.75. This difference of 2.88 points on the PI-NRS was statistically significant (p < 0.05). Participants also reported the intensity of their worst pain from cramps during their most recent menstrual cycle. During the menstrual cycles when the first blood samples were drawn, participants’ worst pain was on average 8.38. During the menstrual cycles of the second blood draws, in which the acupoint injection of phylloquinone occurred, participants’ worst pain was on average 4.50. Additionally, phylloquinone concentrations were negatively correlated with pain intensity of worst cramps during most recent menstrual cycle (r = -0.59), with higher pain ratings associated with lower concentrations of phylloquinone (see Figure 1). Because pain intensity varies with time and blood was sampled at various time points, these findings are suggestive. Confirmation of these findings using a larger clinical trial with multiple, timed blood samples is required before conclusions are drawn regarding the absolute effectiveness of phylloquinone in reducing menstrual pain.

Figure 1.

Correlation between plasma phylloquinone concentrations and pain intensity during last menstrual cycle

Discussion

Our study findings provide evidence of increased concentration of phylloquinone when injected at the acupuncture point SP6. Vitamin K is typically studied in the context of blood clotting and bone health though recent emerging research suggests that vitamin K may have other roles, including reproductive health [9]. Vitamin K therapy may decrease the length of prolonged menstrual flow as a result of its action on prothrombin, a vitamin K-dependent coagulation protein produced in the liver [17]. The association between the liver and menstrual flow is consistent with traditional Chinese medicine (TCM) theory which sees the liver as a key organ in the physiology of menstruation critical in moving blood and qi. From a TCM perspective, stagnation of liver qi or liver blood are major causes of painful periods [5]. The acupuncture point SP6 is commonly used for menstrual conditions because it is a crossing point of the liver, spleen, and kidney channels, which are important in creating, storing, and moving blood [5]. Alternatively, vitamin K is being explored for its potential role in reducing pro-inflammatory cytokines [8], which may also contribute to the beneficial effects.

Our study is limited by the small sample size of only four women, the lack of a comparison group. Furthermore, the study design precludes drawing any causal inferences from our data since pain intensity varies with time and blood samples were drawn at variable intervals before and after acupoint injection. Nonetheless, our finding of high plasma phylloquinone concentrations after intramuscular injection of vitamin K warrants additional research on the extent of synergistic action with site of injection, as suggested by prior studies of acupoint injection [18, 19]. In addition our finding that phylloquinone concentrations and pain intensity are correlated warrants additional research to explore possible mechanisms of acupoint injection of vitamin K and dysmenorrhea, including the association between vitamin K deficiency and dysmenorrhea, the role of vitamin K and prostaglandins, inflammation and pain.